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Analytical Performance of Accelerator Mass Spectrometry and Liquid Scintillation Counting for Detection of 14 C-Labeled Atrazine Metabolites in Human Urine
Abstract
Accelerator mass spectrometry (AMS) has been applied to the detection of 14C-labeled urinary metabolites of the triazine herbicide, atrazine, and the analytical performance of AMS has been directly compared to that of liquid scintillation counting (LSC). Ten human subjects were given a dermal dose of 14C-labeled atrazine over 24 h, and urine from the subjects was collected over a 7-day period. Concentrations of 14C in the samples have been determined by AMS and LSC and range from 1.8 fmol/mL to 4.3 pmol/mL. Data from these two methods have a correlation coefficient of 0.998 for a linear plot of the entire sample set. Accelerator mass spectrometry provides superior concentration (2.2 vs 27 fmol/mL) and mass (5.5 vs 54,000 amol) detection limits relative to those of LSC for these samples. The precision of the data provided by AMS for low-level samples is 1.7%, and the day-to-day reproducibility of the AMS measurements is 3.9%. Factors limiting AMS detection limits for these samples and ways in which these can be improved are examined.
... The extraordinarily high sensitivity of AMS for 14 C labeled materials is based on its detection method of counting individual atoms of the isotope as the free carbon rather than waiting for radioactive decay. AMS precisely quantifies the elevation in 14 C/C above the low natural background abundance in the biosphere ( 14 C/C ¼ 1.2 Â 10 À12 ) and is approximately 100 000Â more sensitive than liquid scintillation counting (Gilman et al., 1998;Vogel and Love, 2005). AMS was utilized to examine the pharmacokinetics of an occupational or environmental dermal exposure to permethrin. ...
... Aliquots of saliva and urine samples were submitted for carbon analysis. For measurement, volume of 5.0 ll was added to a silica support (Chromsorb W/AW 80% 100 mesh; Carlo Erba/ Fisons Instruments, Valencia, California) and analyzed using a Carlo Erba model 1500 NCS (Valencia, California) elemental analyzer (Gilman et al., 1998). Extensive experience has shown that the carbon content of plasma samples is consistent and averages 4.2% so serum samples were not analyzed for carbon content (Buchholz et al., 1999a;Vogel and Love, 2005). ...
Impregnating military uniforms and outdoor clothing with the insecticide permethrin is an approach to reduce exposure to insect borne diseases and to repel pests and disease vectors such as mosquitos and sandflies, but the practice exposes wearers to prolonged dermal exposure to the pesticide. Key metabolite(s) from a low dose dermal exposure of permethrin were identified using accelerator mass spectrometry. Metabolite standards were synthesized and a high performance liquide chromatography (HPLC) elution protocol to separate individual metabolites in urine was developed. Six human subjects were exposed dermally on the forearm to 25 mg of permethrin containing 1.0 mCi of 14 C for 8 h. Blood, saliva and urine samples were taken for 7d. Absorption/elimination rates and metabolite concentrations varied by individual. Average absorption was 0.2% of the dose. Serum concentrations rose until 12-24 h postdermal application then rapidly declined reaching predose levels by 72 h. Maximum saliva excretion occurred 6 h postdosing. The maximum urinary excretion rate occurred during 12-24 h; average elimination half-life was 56 h. 3-Phenoxybenzyl alcohol glucuronide was the most abundant metabolite identified when analyzing elution fractions, but most of the radioactivity was in still more polar fractions suggesting extensive degradative metabolism and for which there were no standards. Analyses of archived urine samples with the ultra performance liquid chromatography-accelerator mass spectrometry-mass spectrometry (UPLC-AMS-MS) system isolated a distinct polar metabolite but it was much diminished from the previous analyses a decade earlier.
... For many years, triazine herbicides have been used in the agriculture industry for corn and sugarcane fields and have shown that herbicides are more persistent in the water body. The environmental fate of residues was found in a number of authors as listed in water [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], in soil [16][17][18][19][20][21][22][23][24], and in plants determined [21][22][23][24][25], blood/plasma [26][27][28][29][30][31], saliva/urine [9,10], milk [5], mode of action, metabolism, photostability [32], decontamination [33], bioaccumulation [34], and mammalian toxicity have been widely described by different researchers [35][36][37][38][39][40]. ...
... For many years, triazine herbicides have been used in the agriculture industry for corn and sugarcane fields and have shown that herbicides are more persistent in the water body. The environmental fate of residues was found in a number of authors as listed in water [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], in soil [16][17][18][19][20][21][22][23][24], and in plants determined [21][22][23][24][25], blood/plasma [26][27][28][29][30][31], saliva/urine [9,10], milk [5], mode of action, metabolism, photostability [32], decontamination [33], bioaccumulation [34], and mammalian toxicity have been widely described by different researchers [35][36][37][38][39][40]. ...
A new multi-residue method using gas chromatography–mass spectrometry electron ionization selective ion monitoring mode (SIM) has been developed for the simultaneous determination of eight 1,3,5-triazine herbicides such as 1,3,5-triazine-2,4-diamine (atrazine), ametryn, prometryn, propazine, terbuthylazine, terbutryn, and simazine simetryn in water and soil samples. Quantification is done using lindane (gamma benzene hexachloride) as an internal standard. A specific Capillary DB-Wax column of 30 m length, 0.32 mm internal diameter, and 0.25 µm film thickness is used for the separation of eight 1,3,5-triazine-2,4-diamine. The method was applied for the determination of residues in groundwater and soil samples. The lowest detection limit by GC-MS-SIM (selective ion monitoring mode) is 0.1 pg/mL. Recovery in water samples is in the range of 93–103%, and in soil samples, 91–102% for different individual compounds. Forty-five groundwater and soil samples were collected in and around Kancheepuram district in Tamilnadu (India), and they were analyzed for the respective residues. A detailed discussion of the GC-MS analysis results has been presented.
... The extraordinarily high sensitivity of AMS for 14 C labeled materials is based on its detection method of counting individual atoms of the isotope as the free carbon rather than waiting for radioactive decay. AMS precisely quantifies the elevation in 14 C/C above the low natural background abundance in the biosphere ( 14 C/C ¼ 1.2 Â 10 À12 ) and is approximately 100 000Â more sensitive than liquid scintillation counting (Gilman et al., 1998;Vogel and Love, 2005). AMS was utilized to examine the pharmacokinetics of an occupational or environmental dermal exposure to permethrin. ...
... Aliquots of saliva and urine samples were submitted for carbon analysis. For measurement, volume of 5.0 ll was added to a silica support (Chromsorb W/AW 80% 100 mesh; Carlo Erba/ Fisons Instruments, Valencia, California) and analyzed using a Carlo Erba model 1500 NCS (Valencia, California) elemental analyzer (Gilman et al., 1998). Extensive experience has shown that the carbon content of plasma samples is consistent and averages 4.2% so serum samples were not analyzed for carbon content (Buchholz et al., 1999a;Vogel and Love, 2005). ...
Impregnating military uniforms and outdoor clothing with the insecticide permethrin is an approach to reduce exposure to insect borne diseases and to repel pests and disease vectors such as mosquitos and sandflies, but the practice exposes wearers to prolonged dermal exposure to the pesticide. Key metabolite(s) from a low dose dermal exposure of permethrin were identified using accelerator mass spectrometry. Metabolite standards were synthesized and an HPLC elution protocol to separate individual metabolites in urine was developed. Six human subjects were exposed dermally on the forearm to 25 mg of permethrin containing 1.0 µCi of 14C for 8 h. Blood, saliva and urine samples were taken for 7d. Absorption/elimination rates and metabolite concentrations varied by individual. Average absorption was 0.2% of the dose. Serum concentrations rose until 12-24 h post dermal application then rapidly declined reaching predose levels by 72 h. Maximum saliva excretion occurred 6 h post dosing. The maximum urinary excretion rate occurred during 12-24 h; average elimination half-life was 56 h. 3-Phenoxybenzyl alcohol glucuronide was the most abundant metabolite identified when analyzing elution fractions, but most of the radioactivity was in still more polar fractions suggesting extensive degradative metabolism and for which there were no standards. Analyses of archived urine samples with the UPLC-AMS-MS system isolated a distinct polar metabolite but it was much diminished from the previous analyses a decade earlier.
... Currently, due to the providing high sensitivity and specificity, mass spectrometry (MS) based methods are widely employed in metabolomics studies (Royston et al., 1996; Seetharaman et al., 2005; Vorst et al., 2005; Lewen et al., 2006; Pa¨rPa¨r et al., 2006; Rainer et al., 2006;), particularly for trace analytes (e.g. urinary atrazine (Gilman et al., 1998; Beeson et al., 1999; Norrgran et al., 2006; Ross and Filipov, 2006)) detection although other techniques such as high performance liquid chromatography (HPLC) (Norrgran et al., 2006; Pozzebon et al., 2003), gas chromatography (GC) (Perry et al., 2000; Pellizzari et al., 2003) and optical sensors (Nichkova et al., 2003) are also used for urine analysis. Traditionally, tedious sample clean-up and preparation are required before sample analysis in MS, resulting in insufficient throughput and rendering difficulties for fast screening of candidates exposed to atrazine. ...
... Urine analysis is widely performed for metabolomics studies and clinical diagnosis in nowadays. Traditionally , mass spectrometric urine analysis (Gilman et al., 1998; Beeson et al., 1999; Norrgran et al., 2006; Ross and Filipov, 2006 ) requires sample clean-up and separation before detection. Desorption electrospray ionization MS (Takats et al., 2004; Cooks et al., 2006) usually requires no sample pretreatment for various surfaces (Takats et al., 2004; Chen et al., 2005; Cooks et al., 2006). ...
Herbicides such as atrazine are widely used in the biosphere. Urine analysis is usually performed to evaluate the toxicological
effects associated with atrazine exposure. A simple procedure based on the extractive electrospray ionization mass spectrometry
(EESI-MS) method was established to detect atrazine and its metabolites in undiluted raw urine without sample pretreatment.
A 4.3×10−14g atrazine in spiked raw urine was detected and identified by EESI/MS/MS/MS. The detection limit was found to be 0.4fg for
atrazine (m/z 174) and 0.2fg for 2-chloro-4, 6-diamino-S-triazine (DACT) (m/z 129) (S/N=3) in EESI/MS/MS. A linear dynamic
range of 4–5 orders of magnitude (r=0.996) was determined for both atrazine and DACT. A single sample analysis was completed using tandem EESI-MS/MS within
1min, providing a practical convenient method for rapid analysis of trace amounts of targeted metabolites present in complex
matrices. Thus, tandem EESI-MS is potentially useful for previously discovered biomarker detection in multiple applications
such as clinical diagnosis, drug discovery and forensic science.
... producing radiation risks that are less than those from a few days of added 'life' [10]. Such tracer studies have quantified the covalent binding of natural toxins and therapeutics to human macromolecules [11,12]; quantified the biochemistry of low-dose exposures to agricultural chemicals [13] and very low-dose nerve agents [14]; followed physiological doses of vitamins even in childbearing age women [15]; revealed the kinetic relationships between high-and low-dose pharmaceuticals [16]; revealed kinetics and metabolism in pharmaceutical research [17] and even indicated biliary status in week-old neonates [18]. This paper aims to confirm the fundamental accuracy and precision of bioanalytical AMS to better than ±2%, which places AMS in a quantitatively different category from bioanalytical instrumentation for which uncertainties of 15-20% are often acceptable. ...
... FiguRe 4B shows the equivalence of measured 14 C transmissions and those calculated as 13/14-times the individually measured 13 C transmissions (unfilled frequency distribution in FiguRe 4B). Transmission efficiency for the 14 C ions through MICADAS is calculated from 13 C transmission to 0.3% accuracy and 1.1% precision, which produces an unnormalized direct quantitation of the 14 C/ 12 C abundance requiring no reference standard other than the sample's own (or an average biological) 13 C content. Hypotheses as to why this spectrometer design operates in this unusual manner are inappropriate in this article. ...
Accelerator mass spectrometers have an energy acceleration and charge exchange between mass definition stages to destroy molecular isobars and allow single ion counting of long-lived isotopes such as (14)C (t½=5370 years.). 'Low' voltage accelerations to 200 kV allow laboratory-sized accelerator mass spectrometers instruments for bioanalytical quantitation of (14)C to 2-3% precision and accuracy in isolated biochemical fractions. After demonstrating this accuracy and precision for our new accelerator mass spectrometer, we discuss the critical aspects of maintaining quantitative accuracy from the defined biological fraction to the accelerator mass spectrometry quantitation. These aspects include sufficient sample mass for routine rapid sample preparation, isotope dilution to assure this mass, isolation of the carbon from other sample combustion gasses and use of high-efficiency biochemical separations. This review seeks to address a bioanalytical audience, who should know that high accuracy data of physiochemical processes within living human subjects are available, as long as a (14)C quantitation can be made indicative of the physiochemistry of interest.
... AMS is an adaptation of stable isotope ratio mass spectrometry that has been modified for tracing biological pathways using multiple isotopic masses. 7 The strength of AMS lies in its ability for longer lived isotopes that cannot be effectively quantified by decay counting. 7 This is especially useful for measuring 14 C, half-life of 5730 years, in biological samples at extremely low concentrations. ...
We have documented that the herbicide propanil, is immunotoxic in mice and our in vitro tissue culture experiments largely recapitulate the in vivo studies. Laboratory studies on environmental contaminants are the most meaningful when these studies are conducted using concentrations that approximate levels in the environment. Many techniques to measure the distribution and pharmacokinetics (PK) on compounds rely on techniques, such as liquid scintillation counting (LSC) of radio-labeled starting compound require concentrations higher than environmental levels. The aim of this study was to compare tissue PK after exposure to propanil concentrations more relevant to levels of exposure to agricultural workers and the general population to concentrations previously reported for laboratory studies. To this end, we conducted a study to measure propanil distribution in three immune organs, using ultrasensitive accelerator mass spectrometry (AMS). We used two doses; the lower dose modeled levels expected in the environment or long-term occupational exposure to low doses, while the higher dose was to model the effects of an accidental exposure. Our results showed that the distribution and PK profiles from these two different concentrations was markedly different. The profile of the high dose (concentration) exposure was indicative of saturation of the detoxifying capability of the animal. In contrast, at the lower environmentally-relevant concentration, in vivo concentrations of propanil in spleen, liver and blood dropped to a very low level by 720 minutes. In conclusion, these studies highlight the differences in PK of propanil at these two doses, which suggests that the toxicity of this chemical should be re-investigated to obtain better data on toxic effects at doses relevant for humans.
... Human exposure to atrazine or terbuthylazine results in a relatively fast urinary excretion of the parent compounds and their dealkylated and conjugated metabolites (104,105). The half-life of atrazine, for example, is only 24-31 h (106,107). The parent compound is, therefore, generally detected only in minor amounts in the urine, usually after continuous occupational exposure. ...
This review article provides a summary of the studies relying on oxidative stress biomarkers (lipid peroxidation and antioxidant enzymes in particular) to investigate the effects of atrazine and terbuthylazine exposure in experimental animals and humans published since 2010. In general, experimental animals showed that atrazine and terbuthylazine exposure mostly affected their antioxidant defences and, to a lesser extent, lipid peroxidation, but the effects varied by the species, sex, age, herbicide concentration, and duration of exposure. Most of the studies involved aquatic organisms as useful and sensitive bio-indicators of environmental pollution and important part of the food chain. In laboratory mice and rats changes in oxidative stress markers were visible only with exposure to high doses of atrazine. Recently, our group reported that low-dose terbuthylazine could also induce oxidative stress in Wistar rats. It is evident that any experimental assessment of pesticide toxic effects should take into account a combination of several oxidative stress and antioxidant defence biomarkers in various tissues and cell compartments. The identified effects in experimental models should then be complemented and validated by epidemiological studies. This is important if we wish to understand the impact of pesticides on human health and to establish safe limits.
... Unlike LSC, which counts the decay events of radioisotopes, AMS counts the individual 14 C atoms which makes this method 10 5 to 10 6 more sensitive than LSC. In addition validation studies comparing AMS to LSC for analysis of 14 C-labeled drugs in diverse biological matrices demonstrated that AMS is an ultrasensitive and robust method for the detection of 14 C in these matrices (Garner et al., 2000;Gilman et al., 1998). The sensitivity of AMS allows for lower dosing, also known as a microdose or microtrace, which may be less than 1% of the therapeutic dose ( 100 lg and 100 nCi 14 C radioactivity). ...
Human radiolabeled mass balance studies are performed to obtain information about the absorption, distribution, metabolism, and excretion of a drug in development. The main goals are to determine the route of elimination and major metabolic pathways. This review provides an overview of the current regulatory guidelines concerning human radiolabeled mass balance studies and discusses scientific trends seen in the last decade with a focus on mass balance studies of anticancer drugs. This paper also provides an overview of mass balance studies of anticancer agents that were executed in the last 10 years.
... The prerequisite to combining AMS and LSC results into one PK data set is a linear relation between both methods. AMS has been compared with LSC, and has shown a good correlation [18,19]. However, either LSC results were extrapolated to the range of the AMS samples, or the LSC samples were diluted before analysis on the AMS. ...
... The 14 C determinations were made at the Center for Accelerator Mass Spectrometry at Lawrence Livermore National Laboratories (Livermore, CA) as described previously [19]. Briefly, plasma samples were thawed at room temperature, vortexed and centrifuged in order to obtain a homogeneous liquid sample. ...
... Early biological AMS investigations in the early 1990s were used to estimate the turnover times of elastin in cadaver lung tissues [7] and genotoxic reactive metabolites of environmental origin that were undetectable in circulation [8,9]. Since then, AMS has been used to investigate low-dose exposure to agricultural chemicals [10], identify the biomolecular targets of 14 C-organophosphate toxins [11], quantify tissue turnover rates [12][13][14], determine the metabolic fate of vitamins and essential dietary chemicals in women of child-bearing age [15][16][17] and assess the biliary status of week-old neonates using labeled bile acids [18,19]. ...
Radiocarbon (¹⁴C) is produced continuously in the upper atmosphere and incorporated into biological systems through photosynthesis. Incorporation of ¹⁴C ceases at death, hence by measuring natural levels of radiocarbon in deceased organic materials the age of the objects (e.g. vegetation, wood and historical artifact, among others) can be determined. Early carbon dating determinations (starting in the late 1940s) relied on monitoring the small levels of radioactivity decay energy emerging from the sample. In the late 1970s nuclear physicists in collaboration with archeologists developed a far more sensitive and efficient measuring technique known as accelerator mass spectrometry (AMS), which can identify individual carbon atoms. The technology was extended to biomedical research in the early 1990s. Here it had an immediate impact on in vivo human research across diverse areas such as carcinogenesis and human nutrition. The same carbon dating technology now supports human ¹⁴C-tracer metabolism studies conducted as part of early drug development. The high sensitivity of AMS enables scientists to explore new drug candidates safely in any human subpopulation at very low chemical (microdosing) and radioactive doses (microtracing). A ‘big physics’ tool originally developed for dating archeological artifacts is now benefiting modern drug development in unanticipated ways. The full benefits of the sensitive measurement technology were impeded by the scale and operational complexity of the early instruments, but this situation has changed significantly over the last decade after the emergence of purpose-built biomedical instruments. The evolving relationship between radiocarbon, AMS, and modern drug development is discussed in this review.
... [ 14 C]atrazine, atrazine mercapturate elimination in urine peaked at 12 h after dosing and decreased to nearly baseline levels by 24 h (Brady et al., 1998). In dermally dosed human volunteers, atrazine, measured as 14 C-labeled urinary metabolites, had a e t 1/2 of 24-28 h (Gilman et al., 1998). Human or primate data on cyanazine metabolism were not found. ...
Metabolites and/or parent compounds of the herbicides atrazine, alachlor, metolachlor, cyanazine and the 2-ethylhexyl ester of 2,4-dichlorophenoxyacetic acid (2,4-D were measured in the urine of 15 custom applicators who each provided from five to seven 24 h urine samples during a 6 week period (n = 87). Each applicator provided a pre-season urine sample and a reference population (n = 46) provided first-morning urine samples. Urinary biomarkers were measured by either immunoassay or gas chromatography. During the spraying season, the geometric mean amount of alachlor mercapturate equivalents (eq.), atrazine eq., 2,4-D and metolachlor mercapturate eq. excreted in 24 h was 17, 19, 110 and 22 nmol, respectively. Mixed-effect models were used to determine predictors of the amount of atrazine eq. and 2,4-D excreted in 24 h. The specific days of herbicide spraying associated with increased biomarker excretion varied for the two analytes, and included one or more days prior to urine collection. This confirms the importance of collecting covariate information on day(s) most relevant to the biomarker of interest. The within-worker variance component, expressed as a geometric standard deviation ( w GSD range: 2.5-2.9), was substantially larger than the between-worker component ( B GSD) range: 1.3-1.5) for the modeled biomarkers. Alachlor mercapturate eq. and metolachlor mercapturate eq. were detected in more than hall of the applicator pre-season urine samples. All biomarkers were detected infrequently in the reference population. Evaluation of non-spray exposure determinants was limited by inclusion of prior day spraying, adjustment for time and the small sample size.
... The low background of 14 C in the environment provides an unambiguous baseline for clearly identifying 14 C-enriched fractions collected from chromatographic systems. 28,29 Furthermore, since quantitation is based upon an intrinsic property of the molecule, i.e., the label, values are more quantitative than from methods whose accuracy is tied to molecular structure or matrix. ...
Vegetables and fruits provide an array of microchemicals in the form of vitamins and secondary metabolites (phytochemicals) that may lower the risk of chronic disease. Tracing these phytochemicals at physiologic concentrations has been hindered by a lack of quantitative sensitivity for chemically equivalent tracers that could be used safely in healthy people. Accelerator mass spectrometry is a relatively new technique that provides the necessary sensitivity (in attomoles) and measurement precision (<3%) towards 14Clabeled phytochemicals for detailed kinetic studies in humans at dietary levels.
... The potential importance of AMS to biomedical tracing was one driver for the development of smaller spectrometers in the following decade [5,6] while the quantitative potential of AMS for determining rare biomolecular binding events was further demonstrated by our colleagues in animal models and in human volunteers [7,8]. Quantification of the pharmacokinetics and metabolism of nutrients or occupational toxins directly in human subjects at realistic doses also broadened the potential of AMS measurements to clinical sciences [9,10]. However, a prediction by this first author at the fourth AMS conference (Paris, 1990) that there would be a majority of AMS systems doing biomedical measurements within a decade has proven wildly inaccurate [11]. ...
... Usporedili su rezultate analize akceleracijskom spektrometrijom masa s rezultatima mjerenja scintilacijskim brojačem kako bi istražili prikladnost ovih dviju tehnika za određivanje ukupnih metabolita atrazina, ali i mogućnost detekcije pojedinih spojeva odijeljenih tekućinskom kromatografijom visoke djelotvornosti. Masene koncentracije atrazina obilježenog izotopom 14 C određene u urinu tim vrlo osjetljivim i ponovljivim metodama bile su razine fmol mL -1 (60). ...
Biološki Monitoring Izloženosti Ljudi Triazinskim Herbicidima Analizom Metabolita u Urinu
Triazinski herbicidi jedni su od najčešće rabljenih herbicida. Sudbina triazinskih herbicida nakon dospijevanja u okoliš ovisi o njihovu kretanju u zraku, vodi i tlu, kao i o brzini kojom se razgrađuju ili transformiraju. Triazinski spojevi mogu se transformirati reakcijom s vodom, djelovanjem mikroorganizama i utjecajem Sunčeva svjetla. Široka i često nekontrolirana primjena triazinskih herbicida tijekom nekoliko proteklih desetljeća rezultirala je kontaminacijom pitke vode i hrane postajući rizik za opću populaciju. Metabolizam i djelovanje triazinskih herbicida proučavani su na pokusnim životinjama i u uvjetima in vitro , dok je o njihovu metabolizmu i izlučivanju kod ljudi malo objavljenih radova. Djelovanju triazinskih herbicida osobito su izloženi radnici zaposleni u njihovoj proizvodnji i primjeni. Izloženost ljudi prati se i dokazuje određivanjem izvornih spojeva i njihovih metabolita u urinu. Kako se triazinski spojevi u urinu profesionalno izloženih ljudi pojavljuju u niskim koncentracijama, za njihovo dokazivanje i praćenje profesionalne izloženosti potrebno je primijeniti analitičke metode visoke djelotvornosti, osjetljivosti i selektivnosti. U ovom radu opisani su struktura i svojstva simetričnih triazinskih herbicida, njihov metabolizam i djelovanje u organizmu čovjeka i životinja te razine metabolita tih spojeva određene u urinu ispitanika iz profesionalno izložene populacije.
... Accelerator mass spectrometry is now increasingly used in clinical studies with a microdose of 14 C-labeled drug tracing a normal clinical dose (unlabeled); this has been termed 'microtracing' [11,12]. This high-sensitivity microtracing has been the only practical analytical solution for certain tissue-or fluid-limited analyses (cells, biopsies and pediatrics), when radiolysis or dosimetry issues are of concern, and for the study of low systemic bioavailability routes of delivery (transdermal, ocular and pulmonary) [13,14]. One microtracing design gaining wide industrial adoption is to measure human clearance and absolute bioavailability by the simultaneous dosing of an intravenous microdose of 14 C-labeled drug with a clinical dose administered by an alternative intended route (e.g., oral) [5,12,15]. ...
Quantitative assessment of metabolites of drug candidates in early-phase clinical development presents an analytical challenge when methods, standards and assays are not yet available. Radioisotopic labeling, principally with radiocarbon ((14)C), is the preferred method for discovering and quantifying the absolute yields of metabolites in the absence of reference material or a priori knowledge of the human metabolism. However, the detection of (14)C is inefficient by decay counting methods and, as a result, high radiological human (14)C-doses had been needed to assure sensitive detection of metabolites over time. High radiological doses and the associated costs have been a major obstacle to the routine (and early) use of (14)C despite the recognized advantages of a (14)C-tracer for quantifying drug metabolism and disposition. Accelerator mass spectrometry eliminates this long-standing problem by reducing radioactivity levels while delivering matrix-independent quantitation to attomole levels of sensitivity in small samples or fractionated isolates. Accelerator mass spectrometry and trace (14)C-labeled drugs are now used to obtain early insights into the human metabolism of a drug candidate in ways that were not previously practical. With this article we describe some of our empirically based approaches for regualted bioanalysis and offer perspectives on current applications and opportunities for the future.
... Although the possibility for AMS to biomedical research was suggested shortly after its development, its use was not explored in earnest until the late 1980's, pioneered by efforts at the Lawrence Livermore National Lab [1,[3][4][5][6][7]. Some of the earliest AMS applications laid the foundation for pharmaceutical and expanded research into biomedical by quantifying concentrations of metabolites in humans, primarily activated metabolites of environmental compounds that form genotoxic macromolecular adducts [8][9][10][11], agricultural chemicals that chronically expose workers at low levels [12][13], and vitamins at physiologically relevant doses [14][15][16]. Prior to AMS, such metabolites and their molecular interactions were only quantified at well above relevant biological concentrations due to the lack of analytical sensitivity. ...
Accelerator Mass Spectrometry is an established technology whose essentiality extends beyond simply a better detector for radiolabeled molecules. Attomole sensitivity reduces radioisotope exposures in clinical subjects to the point that no population need be excluded from clinical study. Insights in human physiochemistry are enabled by the quantitative recovery of simplified AMS processes that provide biological concentrations of all labeled metabolites and total compound related material at non-saturating levels. In this paper, we review some of the exploratory applications of AMS (14)C in toxicological, nutritional, and pharmacological research. This body of research addresses the human physiochemistry of important compounds in their own right, but also serves as examples of the analytical methods and clinical practices that are available for studying low dose physiochemistry of candidate therapeutic compounds, helping to broaden the knowledge base of AMS application in pharmaceutical research.
... 3-ethylaniline Mice [140] Acrylamide Mice [141,148] Aflatoxin B(1) Rats, humans [47,123] Atrazine Humans [4,124] Benzene Mice [120,127,131,134] Benzopyrene Not available [128] Dibromomethane Mice, rats [144] Dichloromethane Mice, rats [144] Ethylene oxide Rats [149] Isofluorophate Mice [130] MeIQx Rats, humans [43,45,[48][49][50]118,119] MTBE Mice [136,145] Nicotine Mice [52] Nitrobenzene Mice [53,132] Ochratoxin A Rats [135,138] Ortho-phenylphenol Rats [125] Oxaliplatin Human breast and bladder cancer cells [143] Tamoxifen Mice, rats, humans [121,122,129,133,142] Tert-butyl alcohol Mice [145] Toremifene Rats [122] ...
Accelerator mass spectrometry is a detection platform with exceptional sensitivity compared with other bioanalytical platforms. Accelerator mass spectrometry (AMS) is widely used in archeology for radiocarbon dating applications. Early exploration of the biological and pharmaceutical applications of AMS began in the early 1990s. AMS has since demonstrated unique problem-solving ability in nutrition science, toxicology and pharmacology. AMS has also enabled the development of new applications, such as Phase 0 microdosing. Recent development of AMS-enabled applications has transformed this novelty research instrument to a valuable tool within the pharmaceutical industry. Although there is now greater awareness of AMS technology, recognition and appreciation of the range of AMS-enabled applications is still lacking, including study-design strategies. This review aims to provide further insight into the wide range of AMS-enabled applications. Examples of studies conducted over the past two decades will be presented, as well as prospects for the future of AMS.
... The tributyrin had a background of 0.24 Fm (fraction modern) ± 0.008 Fm. It should be noted that we have switched to the tributyrin produced by MP Biomedicals (Solon, OH) since it has a much lower background of about 0.1 Fm [13]. 48.66 mg of carbon from the tributryin/methanol carrier (990 ml of solution) was added to 10 μl of the unknown brain MD, which contained less than 100 μg of carbon. ...
Bioactive compounds in botanicals may be beneficial in preventing age-related neurodegenerative diseases, but for many compounds conventional methods may be inadequate to detect if these compounds cross the blood brain barrier or to track the pharmacokinetics in the brain. By combining a number of unique technologies it has been possible to utilize the power of AMS to study the pharmacokinetics of bioactive compounds in the brain at very low concentrations. (14)C-labeled compounds can be biosynthesized by plant cell suspension cultures co-incubated with radioisotopically-labeled sucrose and isolated and separated into a series of bioactive fractions.To study the pharmacokinetics and tissue distribution of (14)C labeled plant polyphenols, rats were implanted with jugular catheters, subcutaneous ultrafiltration probes and brain microdialysis probes. Labeled fractions were dosed orally. Interstitial fluid (ISF) and brain microdialysate samples were taken in tandem with blood samples. It was often possible to determine (14)C in blood and ISF with a β-counter. However, brain microdialysate samples (14)C levels on the order of 10(7) atoms/sample required AMS technology. The Brain Microdialysate(AUC)/Serum(AUC) ranged from .021- to .029, with the higher values for the glycoside fractions. By using AMS in combination with traditional methods, it is possible to study uptake by blood, distribution to ISF and determine the amount of a dose which can reach the brain and follow the pharmacokinetics in the brain.
... One study found that AMS provides superior mass (5 . 5 v. 54 000 amol) detection limits relative to LSC for a particular set of samples (Gilman et al. 1998). This improved detection efficiency makes AMS ideal for studies requiring high sensitivity. ...
Accelerator mass spectrometry (AMS) is an ultrasensitive analytical technique for measuring rare nuclides such as 14C, 26Al and 41Ca. The low detection limit and wide dynamic range of AMS allow long-term and highly sensitive tracer studies in nutrition that cannot be performed with other methods. The present paper is intended to provide a description of AMS to the interested nutritionist and present proven applications. AMS is compared to liquid scintillation counting and stable isotope MS. A description of common AMS methodology is presented that consists of determining the dose, preparing the sample, diluting the sample (if necessary), and measuring the sample. Applications include Ca metabolism, Al uptake from the environment, dietary intake of carcinogens, fat meta-bolism and folate metabolism. Throughout this discussion the experimental advantages (small doses that pose no health risk, extremely long experimental lifetime, small sample sizes and high sensitivity) made possible by the unique analytical capabilities of AMS are emphasized. The future of AMS is discussed. As the number of AMS centres, instruments, and studies increases, the number of nutritional applications that employ AMS will continue to grow. The coupling of AMS with other analytical techniques (e.g. high performance liquid chromatography) will be developed as access to AMS improves.
... However, there were large variations among farmers in the amount of atrazine applied and acreage treated with atrazine (Table 2). Because atrazine has a presumed relatively short biological half-life (24–28h) (Gilman et al., 1998), only applications occurring one day prior to urine collection were included in the analysis (n=66). Correlation between the duration of application and amount of atrazine applied (r Spearman =0.55, p<0.0001); the duration of application and acreage treated (r Spearman = 0.69, p<0.0001); and acreage treated and amount of atrazine applied (r Spearman = 0.72, p<0.0001) was moderate to high. ...
The aim was to develop quantitative estimates of farmers' pesticide exposure to atrazine and to provide an overview of background levels of selected non-persistent pesticides among corn farmers in a longitudinal molecular epidemiologic study. The study population consisted of 30 Agricultural Health Study farmers from Iowa and 10 non-farming controls. Farmers completed daily and weekly diaries from March to November in 2002 and 2003 on pesticide use and other exposure determinants. Urine samples were collected at 10 time points relative to atrazine application and other farming activities. Pesticide exposure was assessed using urinary metabolites and diaries. The analytical limit of detection (LOD) ranged between 0.1 and 0.2 microg/l for all pesticide analytes except for isazaphos (1.5 microg/l) and diazinon (0.7 microg/l). Farmers had higher geometric mean urinary atrazine mercapturate (AZM) values than controls during planting (1.1 vs <LOD microg/g creatinine; P<0.05). AZM levels among farmers were significantly related to the amount of atrazine applied (P=0.015). Interestingly, farmers had a larger proportion of samples above the LOD than controls even after exclusion of observations with an atrazine application within 7 days before urine collection (38% vs 6%, P<0.0001). A similar pattern was observed for 2,4-D and acetochlor (92% vs 47%, P<0.0001 and 45% vs 4%, P<0.0001, respectively). Urinary AZM levels in farmers were largely driven by recent application of atrazine. Therefore, the amount of atrazine applied is likely to provide valid surrogates of atrazine exposure in epidemiologic studies. Elevated background levels of non-persistent pesticides, especially 2,4-D, indicate importance in epidemiologic studies of capturing pesticide exposures that might not be directly related to the actual application.
... Reports on the use of MS as a routine detector for trace analysis of pesticides in the regulatory area (107) and development of guidelines for detection, confirmation, and quantification by MS (108) were published. Accelerator MS and liquid scintillation counting were applied to the detection of l4 C-labeled urinary metabolites of atrazine, and a mass LOD of 5.5 amol was reported (109). Dibutyltin, tributyltin, diphenyltin, and triphenyltin residues in sediments were determined and speciated by HPLC coupled with atmospheric pressure ionization MS and inductively coupled plasma MS (110). ...
Accelerator Mass Spectrometry (AMS) and the Full Combustion (FC) method followed by isotope separation and liquid scintillation counting (LSC) have been applied for measuring the produced and accumulated ¹⁴C in the thermal column of the decommissioned VVR-S reactor in NIPNE Bucharest. The FC method has also been used to measure the tritium (³H) deposition in the reactor column. The two employed methods are complementary and during this study they were the subjects of a cross comparison exercise.
Measurements of the concentrations of radioactive or stable isotopes in body fluids (e.g., blood, plasma), excreta (e.g., urine, breath, feces, saliva) or biopsied tissue after in vivo administration constituted a major part of nuclear medicine in the early years of the specialty (roughly, the third quarter of the 20th Century). This era was mostly research-oriented and pre-dinical. Yet, clinical applications of in vitro tests were developing for the purposes of recognizing problems of gastrointestinal absorption of minerals, vitamins and energy constituents of the diet and the fate of elements or compounds after enterai or parenteral administration. Amounts and kinetics of body constituents were evaluated in ways which were, and in various cases still are, achievable only, or best, with the isotopic tracers. Another important facet of radiotracer use was the development of radioimmunoassay, by which minute concentrations of proteins (especially hormones) in body fluids would be measured with much benefit to clinical medicine. Autoradiography of uptake of weak beta emitters into isolated cells after in vivo or in vitro exposure also developed in this period, as did initial work with in vitro blood cell tagging for volume assays and for imaging.
The use of radioactivity is a well-established approach to generate data from a wide range of drug metabolism and pharmacokinetic (DMPK) activities, including absorption, distribution, metabolism, and excretion (ADME) studies, investigations of enzymology and enzyme kinetics, and tissue distribution studies in toxicology species. A variety of methodologies is available for the detection and quantitation of radioactive components, comprising a variety of off-line and online approaches. Off-line techniques involve the fractionation of metabolism samples into either scintillation vials or microtiter plates followed by radiochemical analysis of the collected fractions. Online approaches employ radioflow cells, which utilize homogeneous detection, involving the addition of a liquid scintillant, or heterogeneous detection, where solid scintillants are used. In addition, accelerator mass spectrometry (AMS), which detects individual isotope ions, represents an alternative approach to the quantitation of radioactive material. The choice of approach is a balance between sensitivity and considerations of time and cost. In terms of sensitivity, off-line approaches generally offer an improvement over online techniques, although stop-flow approaches can enhance online detection, while AMS provides a dramatic further increase in sensitivity. However, online approaches typically offer higher throughput than off-line techniques, with AMS being extremely slow and costly. For these reasons, case-by-case considerations of the optimal detection approach are necessary, based on an understanding of the capabilities of the various methodologies and the necessary data to address the relevant ADME issues.
Keywords:
radiochemical detection;
metabolites;
quantitation;
accelerator mass spectrometry
The dermal route is a significant pathway for potential exposure to pesticides used in agriculture. The estimation of dermal absorption from in vivo, in vitro, animal, and mathematical models is a key component of occupational risk characterization. The classic 10 steps of percutaneous penetration are expanded to 15 factors, which will be outlined briefly. These factors should be considered when designing or interpreting dermal absorption studies.
Relevance of AMS to Drug MetabolismIntroduction to AMSFundamentals of AMS InstrumentsSample Definition and InterfacesAMS QuantitationLC–AMS Analysis of Drug MetabolitesComparative Resolution of Fraction LC MeasurementsQuantitative Extraction and RecoveryLC–AMS Background and SensitivityClinical Aspects of AMS Metabolite StudiesAMS Analysis of Reactive MetabolitesSpecies Metabolite ComparisonNew Metabolic Studies Enabled by AMSConclusions
References
The goal of this thesis was to advance an implantable drug delivery MEMS (MicroElectroMechanical Systems) device developed in our laboratory. This device was designed to locally deliver multiple substances in complex release profiles in order to maximize the effectiveness of drug therapies. It consists of an array of microreservoirs etched into a silicon substrate. Different types and dosages of drugs can be contained in these reservoirs capped by thin gold membranes. The drug release is achieved by the application of a small anodic potential on the gold membrane in a chloride containing medium (such as the body fluid). The gold membrane will corrode and disintegrate so that the drug contained within the reservoir is free to diffuse into the surrounding medium. Previous researchers have demonstrated in vitro and in vivo release of tracer molecules as well as a radiolabled chemotherapeutic agent (carmustine, or BCNU) from the device. However, systematic characterization of the mechanical and electrochemical behavior of gold membranes on the drug delivery device was necessary in order to achieve more reliable device performance and to demonstrate efficacy of BCNU delivered from the MEMS device against an experimental tumor model. A bulge test apparatus was constructed to characterize the mechanical properties of gold membranes. Uniform pressure was applied from underneath the gold membrane and the membrane deflection was measured using optical interferometry. Analyzing the deflection and pressure data allowed extraction of the elastic modulus and residual stress of the gold membrane.
The skin may be an unintended route for localized and systemic exposure to nanoparticles released during the manufacture, use, and disposal of nanomaterials. Percutaneous absorption is a dynamic process and there are many components with which a penetrant interacts before possibly gaining systemic access. Here, the classic ten steps of percutaneous penetration are expanded to 15 perspectives. These are issues considered from the perspective of nanoparticles and the potential risks of dermal exposure. Although much remains to be done in the field of dermatotoxicology of nanomaterials, uncertainties regarding the impact of nanoscale materials would be helped by greater consistency in methodologies used to assess health risks. Without extensive validation of highly standardized and well-controlled test systems, in vitro data alone cannot be used in place of in vivo data.
IntroductionRadioactivity Detection MethodsAMSIntracavity Optogalvanic SpectroscopySummaryAcknowledgmentsReferences
Bentonite clay was modified using quaternary ammonium cations, viz. phenyltrimethylammonium (PTMA), hexadecyltrimethylammonium (HDTMA), trioctylmethylammonium (TOMA) [100 % of cation exchange capacity of clay], and stearylkonium (SK) [100 % (SK-I) and 250 % (SK-II) of cation exchange capacity of clay]. The organoclays were characterized using X-ray diffraction (XRD), infrared (IR) spectroscopy, and scanning electron microscopy (SEM). Atrazine adsorption on modified clays was studied using a batch method. Bentonite clay was a poor adsorbent of atrazine as 9.4 % adsorption was observed at 1 μg mL−1 atrazine concentration. Modification of clay by PTMA cation did not improve atrazine adsorption capacity. However, atrazine adsorption in HDTMA-, TOMA-, and SK-bentonites varied between 49 and 72.4 % and data fitted well to the Freundlich adsorption isotherm (R > 0.96). Adsorption of atrazine in organoclays was nonlinear and slope (1/n) values were <1. The product of Freundlich adsorption constants, K
f(1/n) in HDTMA-, TOMA-, and SK-I-bentonites was 239.2, 302.4, and 256.6, respectively, while increasing the SK cation loading in the clay (SK-II) decreased atrazine adsorption [K
f(1/n) − 196.4]. Desorption of atrazine from organoclays showed hysteresis and TOMA- and SK-I-bentonites were the best organoclays to retain the adsorbed atrazine. Organoclays showed better atrazine removal from wastewater than an aqueous solution. The synthesized organoclays may find application in soil and water decontamination and as a carrier for atrazine-controlled released formulations.
A major driving force in the development of mass spectrometry is its utility in the discovery and development of drugs in the pharmaceutical industry. With the increased demand for ADME data earlier in the drug discovery process, the sensitivity and versatility of mass spectrometry has proven it to be a reliable and invaluable tool in the pharmacology laboratory. Improved Chromatographic techniques coupled with advances in mass analysis technology have meant that biodistribution and elimination studies can now be developed and implemented in a matter of days rather than weeks or months. Widespread use of electrospray ionization and atmospheric pressure chemical ionization have contributed to ease of use in acquisition of high quality quantitative data necessary for pharmacokinetic studies.
Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical method suitable for detection of sub-nanomolar concentrations of labeled biological substances such as pharmaceuticaldrugs in body fluids. A limiting factor in extending the concentration measurements to the sub-picomolar range is the natural 14C content in living tissues. This can be circumvented by separating the labeled drug from the tissue matrix with, for example, liquid chromatography. The analysis of drugs and their metabolites or endogenous compounds in biological fluids by liquid chromatography is usually complicated and the sample preparation step remains the most serious problem both with regard to losses and degradation of the analyte, and also automation of the analysis. In this article a method for detection and quantification of extremely low concentrations of 14C-labeled biomolecules in biological fluids by AMS is described. The use of a column switched chromatographic system incorporating a restricted-access media (RAM) column allowed the direct injection of untreated human plasma samples, which reduces the total time of analysis and makes automation of the sample preparation step possible. As the separated total drug amount is in the attogram to femtogram region, it is not possible to use a standard AMSsample preparation method, where mg sizes are required. We have utilized a sensitive carboncarrier method where a 14C-deficient compound is added to the HPLC fractions and the composite sample is prepared and analysed by AMS. The method shows remarkable sensitivity, low background values and good linearity, allowing the detection and quantification of a pharmaceuticaldrug in human plasma in the low femtomolar and down to the attomolar concentration range.
This chapter discusses the ultra-sensitive detection of radiolabeled drugs and their metabolites using accelerator mass spectrometry (MS). The current understanding of the metabolism of endogenous and xenobiotic compounds has become possible by the use of molecules labeled with radioisotopes. Usually, the compound under investigation is synthesized so that selected atoms in the molecule are enriched with the desired radioisotope. The resulting radiolabeled compound, which is chemically identical to its nonradioactive counterpart, is then administered to a test system and radioactivity is followed over time. This enables the passage of the compound and its metabolites to be distinguished from the myriad of endogenous substances. The current technique of sample preparation by graphitization is labor intensive. It is important to develop methods to permit samples to be directly inserted into the ion source, thus, eliminating the graphitization process. In contrast to the sample preparation time, accelerator mass spectrometry (AMS) run times per sample can be as short as 100 seconds and still give good precision. AMS is the most sensitive analytic method ever developed because of which a range of studies that were previously impossible, particularly in humans, can now be performed.
Introduction Liquid Chromatography–Accurate Radioisotope Counting–Mass Spectrometry 96-Well Microtiter Plates Containing Solid Scintillant Future Trends in Low-Level Metabolite Characterization in Combination with LC–ARC–MS and 96-Well Plate Approach Accelerator Mass Spectrometry Summary Acknowledgment References
Introduction A Pharmacokinetics Primer Quantitative Bioanalysis During Drug Discovery Quantitative Bioanalysis for Regulatory Submissions Acknowledgments References
Accelerator mass spectrometry (AMS) is an ultrasensitive measure for tracing14C labeled moleculesin vivo or detecting the biomarker for assessment of carcinogenesis. In this review, basic principles, wide applications and new
progresses of14C-bio-AMS are presented. It has been a new advanced tool for measuring the adduction of biologcial molecules with xenobiotics,
including carcinogens, drugs, agrochemicals, nicotine, etc. The successful applications have proven the effectiveness of AMS
to assessing cancer risk, screening drug toxicity and studying nutrients uptake. In particular, AMS is characterized by measuring
xenobiotics at very low dose levels relevant to human environmental exposure. It is sensitive and precise to an attomole (10−18 mole) or less of14C per mg carbon. Although it has some shortcomings, undoubtedly, AMS possesses an evident merit of high sensitivity and will
have widespread applications in the biomedical sciences.
Accelerator mass spectrometry (AMS) is the most sensitive analytical method yet developed for elemental isotope analysis and has a broad range of applications. The measurement of 14C is of most interest to biomedical researchers but few studies have been reported using AMS in drug discovery and development. For biomedical use, 14C is incorporated into organic molecules by either radiosynthesis or biosynthetically and the isotope is used as a surrogate for the distribution of the radiolabelled molecule either in animal or human studies. The majority of users of 14C quantitate the radioactivity using decay counting usually with a liquid scintillation counter (LSC). Our Centre over the past 12 months has been evaluating and validating the use of AMS as an alternative detection method. In vitro spiking studies of human plasma with 14C-Fluconazole, a prescription antifungal drug has demonstrated an excellent correlation between AMS and LSC (correlation coefficient 0.999). Human Phase I clinical studies have been conducted with radioactive doses ranging from 120 Bq (7000 dpm) to 11 kBq (300 nCi) to provide mass balance, plasma concentration and radioactive metabolite profiling data. Limits of detection of 0.00022 Bq 14C-labelled drug/ml plasma have been accurately quantitated in a plasma background of 0.0078 Bq/ml (0.013 dpm/ml in a plasma background of 0.47 dpm/ml or 2.72 pMC in a background of 90.19 pMC).
This paper describes an evaluation of extraction methods of triazine herbicides atrazine, simazine, prometryn, and ametryn and of three chlorodealkylated atrazine metabolites from human urine using diethyl ether and C18 solid-phase extraction (Sep-Pak C18 cartridges). The extracts were analysed by capillary gas chromatography using nitrogen selective and electron capture detectors. At ng ml−1 concentrations the diethyl ether extraction recoveries of atrazine and simazine were almost quantitative (R.S.D. 10%), while the recoveries of other triazines ranged from 58% for deisopropylatrazine to 85% for deethylatrazine (R.S.D. 19–34%). The sensitivity of the method using a nitrogen selective detector ranged from 5 ng ml−1 for atrazine to 30 ng ml−1 for didealkylated atrazine. C18 solid-phase extraction with acetone as eluting solvent resulted in about 100% recoveries of all parent herbicides (R.S.D. 15–21%) with detection limits of 5 ng ml−1 for atrazine and 10 ng ml−1 for other herbicides. Comparably high recoveries of three dealkylated atrazine metabolites (R.S.D. 10–12%) were achieved from acidified urine samples (pH 2–3) only at concentrations lower than 100 ng ml−1. The diethyl ether extraction procedure served to identify occupational exposure to atrazine and simazine. Deethyldeisopropylatrazine was confirmed as the predominant urinary dealkylated metabolite followed by monodealkylated metabolites and unchanged atrazine.
Isotopic labels are a primary tool for tracing chemicals in natural systems. Accelerator mass spectrometry (AMS) quantifies long-lived isotopes that can be used in safe, sensitive and precise biochemical research with human participants. AMS could reduce the use of animals in biochemical research and remove the uncertain extrapolations from animal models to humans. Animal data seldom represent the sort of variability expected in a human population. People, knowingly or not, routinely expose themselves to radiation risks much greater than AMS-based biochemical research that traces μg/kg doses of chemicals containing tens of nCi of 14C for as long as 7 months. AMS is applied to research in toxicology, pharmacology and nutrition.
The ¹⁴C-D-xylose breath test was used at Ullevål University Hospital in the period from 1986 TO 1995 for malabsorption testing. The objective of this retrospective study was to reveal whether patients with chronic alcoholism may have intestinal malabsorption.
The consecutive ¹⁴C-D-xylose breath test database was reviewed and patients with the diagnosis of chronic alcoholism were identified. ¹⁴C-D-xylose breath test results of the alcoholic patients were compared with the results of untreated celiac patients and patient and healthy controls. In the ¹⁴C-D-xylose breath test, ¹⁴C-D-xylose was dissolved in water and given orally after overnight fast. Breath samples were taken at 30-min intervals for 210 min, and ¹⁴CO₂ : ¹²CO₂ ratios were calculated for each time point, presenting a time curve for ¹⁴C-D-xylose absorption. Urine was collected after 210 min and the fraction of the total d-xylose passed was calculated (U%). ¹⁴CO₂ in breath and ¹⁴C-D-xylose in urine were analyzed using liquid scintillation.
Both breath and urine analysis revealed a pattern of malabsorption in alcoholics comparable with untreated celiac patients, with significantly reduced absorption of d-xylose compared with patient and healthy controls.
Alcoholic patients have a significantly reduced ¹⁴C-D-xylose absorption, comparable with untreated celiac patients. This indicates a reduced intestinal function in chronic alcoholism.
Human exposure to atrazine and other triazine herbicides results in urinary excretion of traces of parent compounds and of their metabolites formed by N-dealkylation or conjugation with mercapturic acid. In contrast to N-dealkylated metabolites, which are not compound-specific, the measurement of atrazine mercapturate and unchanged atrazine provides an unambiguous confirmation of exposure to this herbicide. The aim of this study was to investigate the levels of these two compounds in a group of agricultural workers who may be considered representative for typical behaviour and procedures during the atrazine application in Croatia. The spot urine samples were collected at the beginning (samples A) and at the end (samples B) of a working day and 12h after exposure has ended (samples C). Atrazine and atrazine mercapturate were extracted from acidified urine samples (pH 2) with ethyl acetate and the extracts were analysed using high performance liquid chromatography-tandem mass spectrometry with a turbo ion spray (electrospray) ionization interface. The detection limits based on treatment of 2ml urine samples were 0.2ngml(-1) for both analytes. Atrazine was not detected in any of 27 analysed urine samples but traces of atrazine mercapturate were measured in about a third of pre-exposure and in all post-exposure urine samples in mass concentrations ranging from 0.3 to 10.4ngml(-1) (0.3 to 8.0μgg(-1) of creatinine). The metabolite concentrations in B and C group of post-exposure samples were not significantly different. The urinary atrazine mercapturate post-exposure concentrations were comparable to those reported for U.S. farmers engaged in a single field application of atrazine.
5-methoxypsoralen (bergapten) has been used in the treatment of psoriasis and vitiligo, and as a sun tanning accelerator. While low plasma concentrations have previously been detected, there is no data on its dermatopharmacokinetics.
Three rhesus monkeys were used as a model for human skin. [14C]-5-methoxypsoralen, as a parenteral excretion control, was injected in propylene glycol with an activity of 1.12 uCi/ml at a concentration of 80 mcg/ml and urine was collected at 4, 8, 12, 24h, and then daily for a total of 6 days. [14C]-5-methoxypsoralen was then applied topically in acetone with a dose of 1.19 mc (72 mcg) and urine was collected at 4 and 24h and then daily for a total of 7 days. The amount excreted was corrected for the previously determined parenteral excretion kinetics.
Intramuscular [14C]-5-methoxypsoralen had an average of 71.87±7.77% of excretion and percutaneously applied [14C]-5-methoxypsoralen had an average of 58.4±11.8% of excretion.
A high percentage of the administered 5-methoxypsoralen was absorbed. This provides a foundation of methodology to evaluate the efficacy of other delivery vehicles for 5-methoxypsoralen and serves as part of its dermatotoxic profile.
Transdermal hormone application allows delivery of a clinically relevant hormone dose often with fewer systemic side effects than oral formulations. However, transdermal hormone transfer from a dosed individual to naïve interpersonal contact occurs and may cause significant hormone imbalance and adverse effects.
We reviewed PubMed, Medline, and Scopus articles from the years 1950 to 2010 for articles related to transdermal hormone transfer in the setting of in vivo and in vitro human and animal models. We used the following key words: transfer, transdermal, absorption, cutaneous, hormone, estradiol, and testosterone. Unpublished trials were reviewed on the US Food and Drug Administration (FDA) website for product approval.
Data reflecting in vivo transfer of transdermal estradiol and testosterone in man is available from case reports, clinical trials, and FDA product information. While results clearly show that transfer can occur, methods for measuring the effect are not standardized and are thus difficult to compare among positive and negative studies. No in vitro human studies or animal models have been developed to specifically examine transfer potential of transdermal estradiol or testosterone.
It is necessary to consider the mechanism behind transdermal hormone transfer and consider ways to enhance clinical benefits to the dosed individual while minimizing transfer to a naïve interpersonal contact. A detailed discussion of trial comparisons and future optimization methods may help enhance our understanding of the potential for transdermal hormone transfer and encourage development of newer formulations and/or application methods to minimize its occurrence.
In this thesis take-home pesticide exposure among farm families, with an emphasis on herbicides, was investigated. Take-home exposure occurs when a worker unwittingly brings home a substance on his or her clothing or shoes, thereby potentially exposing his or her family. The pesticides investigated were atrazine, metolachlor, acetochlor, alachlor, 2,4-D, glyphosate, and chlorpyrifos; all were applied to either corn or soybean crops. A questionnaire was administered to each family to determine pesticide use and work practices. Urine samples were collected from the participants and dust, wipe and air samples were collected from each home. Atrazine was the agricultural pesticide used most by farmers and more than one agricultural pesticide was applied on most farms. Most farmers changed from their work clothes and shoes in the home, and when they changed outside they usually brought their clothes and shoes inside. Greater than 88% of the air and 74% of the wipe samples were below the limit of detection (LOD). Among the air and wipe samples, chlorpyrifos was detected most frequently in homes. In the dust samples, all the pesticides were detected in greater than 50% of the samples except acetochlor and alachlor, which were detected in less than 30% of the samples. Pesticides in dust samples were detected more often in farm homes except 2,4-D, which was detected in 100 percent of the farm and non-farm home samples. All farms where a pesticide was sprayed had higher levels of that pesticide in dust than both farms that did not spray that pesticide and non-farms, however, only atrazine and metolachlor were significantly higher. For atrazine, acetochlor, metolachlor and 2,4-D, farmers who reported applying the pesticide had significantly higher urinary metabolite levels than non-farmers, farmers who did not apply the pesticide, and farmers who had the pesticide commercially applied. The adjusted geometric mean (GM) level of the urine metabolite of atrazine was significantly higher in fathers, mothers and children from farm households compared to those from non-farm households. Urine metabolites of chlorpyrifos were significantly higher in farm fathers and marginally higher in farm mothers when compared to non-farm fathers and mothers, but metolachlor and glyphosate levels were similar between the two groups. Farm children had significantly higher urinary atrazine and chlorpyrifos levels (p = 0.03 and p = 0.03 respectively) when these pesticides were applied by their fathers prior to sample collection than those of farm children where these pesticides were not recently applied. Estimated doses for the children were calculated for atrazine, metolachlor, chlorpyrifos and glyphosate from urinary concentrations. For all pesticides except glyphosate, the doses from farm children were higher than doses from the non-farm children. The difference was statistically significant for atrazine but only marginally significant for chlorpyrifos and metolachlor. None of the doses exceeded the U.S. Environmental Protection Agency (EPA) chronic reference values for atrazine, metolachlor and glyphosate; however, all of the doses for chlorpyrifos exceeded the EPA chronic population adjusted reference value. The results show that farm homes are contaminated with pesticides and exposure of inhabitants is occurring.
Controlled-release herbicide formulations have significant role in slow release of herbicides under laboratory and field conditions. In the present study kaolin, clay and saw dust were used to sorb the herbicide butachlor to test the slow release formulations of herbicides and their kinetic retention behavior in water. Release of different formulations of butachlor and oxadiazon were also studied. Desorption of oxadiazon formulations OA1 showed higher release (93.98±0.34%) than OA2 (43.73±0.45%), butachlor formulations BH1 exhibited higher release (99.29±0.33%) than BH2 (59.45±0.30%) on 28th day. It was experimentally confirmed that clay and saw dust as a coating material have high adsorption rate (77.2%), found to increase the holding capacity of butachlor rather than kaolin and the release was very slow which may avoid herbicide loss by environmental degradation. Organoclays and saw dust may find application as better sorbents of herbicides than kaolin and this will be a promising formulation in the environmental and economic point of view.
Accelerator mass spectrometry (AMS) is an ultra-sensitive analytical method suitable for the detection of sub-nM concentrations of labeled biological substances such as pharmaceutical drugs in body fluids. A limiting factor in extending the concentration measurements to the sub-pM range is the natural (14)C content in living tissues. This was circumvented by separating the labeled drug from the tissue matrix, using standard high-performance liquid chromatography (HPLC) procedures. As the separated total drug amount is in the few fg range, it is not possible to use a standard AMS sample preparation method, where mg sizes are required. We have utilized a sensitive carbon carrier method where a (14)C-deficient compound is added to the HPLC fractions and the composite sample is prepared and analyzed by AMS. Using 50 microL human blood plasma aliquots, we have demonstrated concentration measurements below 20 fM, containing sub-amol amounts of the labeled drug. The method has the immediate potential of operating in the sub-fM region.
We have developed an isotope dilution high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method for quantifying the urinary metabolites of the pesticides atrazine, malathion, and 2,4-dichlorophenoxyacetic acid (2,4-D). Urine samples are extracted with an organic solvent, and the organic fraction is concentrated. The concentrate is then analyzed using HPLC/MS/MS. The limits of detection for the metabolites are less than 0.5 microgram/L (parts per billion) in 10 mL of urine, with a high degree of accuracy and precision.
The metabolism of 50 μM [2,4,6-14C]atrazine by liver microsomes from ICR strain mice produced metabolites not previously described which account for about 25% of total transformation products, plus 10-14% deethylatrazine and 13-15% deisopropylatrazine, based on the total radioactivity injected into an analytical high-pressure liquid chromatography (HPLC) system. The unknown metabolites were observed by HPLC of products from atrazine incubations with liver microsomes from both untreated mice and mice treated with phenobarbital. Two major unidentified peaks (Unknowns 1 and 2) were isolated and collected by HPLC and characterized by gas chromatography-electron-impact ionization-mass spectrometry. Unknown 1 was an unstable intermediate, likely a mixture of carbinolamines, that decomposed into deethylatrazine, and Unknown 2, a mixture of compounds unseparated by HPLC probably with N-vinyl substituents at the location of the N-isopropyl and N-ethyl groups of the atrazine molecule with M+ at m/z 213. While microsomal oxidation did produce a mixture of trace amounts of breakdown products of atrazine, the method was impractical for the initial goal of preparing pure dealkylated atrazine derivatives for environmental degradation studies.
OUR understanding of how future changes in atmospheric carbon-dioxide concentrations will affect the global climate system arises in part from comparing past changes in climate and CO2. To date, these comparisons have come mainly from ice-core data, which show a strong correlation-between past atmospheric CO2 concentration and polar temperature1. Here we present a new method for reconstructing atmospheric CO2 concentration using the C-13/C-12 ratio (deltaC-13) in mosses and sedges in peat. Our method exploits the fact that, unlike sedges and most other plants, mosses do not possess stomata, and are therefore unable to regulate their uptake of CO2 and water. The deltaC-13 of mosses thus depends on both atmospheric CO2 concentration and available water, and the of sedges from the same peat can be used to remove the water signal. The method provides a resolution of about a decade much higher than is possible from ice cores. We present initial results for the past 14,000 years, which show three sharp increases in atmospheric CO2 concentration: at 12,800 years ago, corresponding to an episode of warming in the North Atlantic region, 10,000 years ago, corresponding to the end of the Younger Dryas cold period; and 4,400 years ago, after which time modern climates were established globally.
Accelerator mass spectrometry (AMS) is used to determine the amount of carcinogen covalently bound to mouse liver DNA (DNA adduct) following very low-level exposure to a 14C-labeled carcinogen. AMS is a highly sensitive method for counting long-lived but rare cosmogenic isotopes. While AMS is a tool of importance in the earth sciences, it has not been applied in biomedical research. The ability of AMS to assay rare isotope concentrations (10Be, 14C, 26Al, 41Ca, and 129I) in microgram amounts suggests that extension to the biomedical sciences is a natural and potentially powerful application of the technology. In this study, the relationship between exposure to low levels of 2-amino-3,8-dimethyl[2-14C]imidazo[4,5-f]quinoxaline and formation of DNA adducts is examined to establish the dynamic range of the technique and the potential sensitivity for biological measurements, as well as to evaluate the relationship between DNA adducts and low-dose carcinogen exposure. Instrument reproducibility in this study is 2%; sensitivity is 1 adduct per 10(11) nucleotides. Formation of adducts is linearly dependent on dose down to an exposure of 500 ng per kg of body weight. With the present measurements, we demonstrate at least 1 order of magnitude improvement over the best adduct detection sensitivity reported to date and 3-5 orders of magnitude improvement over other methods used for adduct measurement. An additional improvement of 2 orders of magnitude in sensitivity is suggested by preliminary experiments to develop bacterial hosts depleted in radiocarbon. Expanded applications involving human subjects, including clinical applications, are now expected because of the great detection sensitivity and small sample size requirements of AMS.
Autoradiography is widely and successfully used to image the distribution of radiolabelled tracer molecules in biological samples. The method is, however, limited in resolution and sensitivity, especially for 14C. Here we describe a new method for imaging 14C-labelled tracers in sections of biological tissue. A highly focused beam of gallium ions bombards the tissue, which is eroded (sputtered) into constituent atoms, molecules and secondary ions. The 14C ions are detected in the secondary beam by the most sensitive method available, namely accelerator mass spectrometry. The specimen is scanned pixel by pixel (1 x 2 microm), generating an image in a manner analogous to scanning electron microscopy. The method can thus be regarded as a specialized form of scanning secondary ion mass spectrometry (SIMS), referred to here as SIAMS (ref. 2). We have used SIAMS to localize the neurotransmitter gamma-aminobutyric acid (GABA) in thin sections of cerebral cortex, and show that it can generate 14C images that are much improved on 14C autoradiography. A scan takes 10-20 min and reveals individual axons, neurons and glial cells at high sensitivity. In principle, the resolution could be increased by up to tenfold, and the method could be extended to some other nuclides.
The triazine herbicide atrazine (2-chloro-4-ethylamino-6-isopropyl-amino-s-triazine) is one of the most used pesticides in North America. Atrazine is principally used for control of certain annual broadleaf and grass weeds, primarily in corn but also in sorghum, sugarcane, and, to a lesser extent, other crops and landscaping. Atrazine is found in many surface and ground waters in North America, and aquatic ecological effects are a possible concern for the regulatory and regulated communities. To address these concerns an expert panel (the Panel) was convened to conduct a comprehensive aquatic ecological risk assessment. This assessment was based on several newly suggested procedures and included exposure and hazard subcomponents as well as the overall risk assessment. The Panel determined that use of probabilistic risk assessment techniques was appropriate. Here, the results of this assessment are presented as a case study for these techniques. The environmental exposure assessment concentrated on monitoring data from Midwestern watersheds, the area of greatest atrazine use in North America. This analysis revealed that atrazine concentrations rarely exceed 20 μg/L in rivers and streams that were the main focus of the aquatic ecological risk assessment. Following storm runoff, biota in lower-order streams may be exposed to pulses of atrazine greater than 20 μg/L, but these exposures are short-lived. The assessment also considered exposures in lakes and reservoirs. The principal data set was developed by the U.S. Geological Survey, which monitored residues in 76 Midwestern reservoirs in 11 states in 1992-1993. Residue concentrations in some reservoirs were similar to those in streams but persisted longer. Atrazine residues were widespread in reservoirs (92% occurrence), and the 90th percentile of this exposure distribution for early June to July was about 5 μg/L. Mathematical simulation models of chemical fate were used to generalize the exposure analysis to other sites and to assess the potential effects of reduction in the application rates. Models were evaluated, modified, and calibrated against available monitoring data to validate that these models could predict atrazine runoff. PRZM-2 overpredicted atrazine concentrations by about an order of magnitude, whereas GLEAMS underpredicted by a factor of 2 to 5. Thus, exposure models were not used to extrapolate to other regions of atrazine use in this assessment. The effects assessment considered both freshwater and saltwater toxicity test results. Phytoplankton were the most sensitive organisms, followed, in decreasing order of sensitivity, by macrophytes, benthic invertebrates, zooplankton, and fish. Atrazine inhibits photophosphorylation but typically does not result in lethality or permanent cell damage in the short term. This characteristic of atrazine required a different model than typically used for understanding the potential impact in aquatic systems, where lethality or nonreversible effects are usually assumed. In addition, recovery of phytoplankton from exposure to 5 to 20 μg/L atrazine was demonstrated. In some mesocosm field experiments, phytoplankton and macrophytes were reduced after atrazine exposures greater than 20 μg/L. However, populations were quickly reestablished, even while atrazine residues persisted in the water. Effects in field studies were judged to be ecologically important only at exposures of 50 μg/L or greater. Mesocosm experiments did not reveal disruption of either ecosystem structure or function at atrazine concentrations typically encountered in the environment (generally 5 μg/L or less). Based on an integration of laboratory bioassay data, field effects studies, and environmental monitoring data from watersheds in high-use areas in the Midwestern United States, the Panel concluded that atrazine does not pose a significant risk to the aquatic environment. Although some inhibitory effects on algae, phytoplankton, or macrophyte production may occur in small streams vulnerable to agricultural runoff, these effects are likely to be transient, and quick recovery of the ecological system is expected. A subset of surface waters, principally small reservoirs in areas with intensive use of atrazine, may be at greater risk of exposure to atrazine. Therefore, it is recommended that site-specific risk assessments be conducted at these sites to assess possible ecological effects in the context of the uses to which these ecosystems are put and the effectiveness and cost-benefit aspect of any risk mitigation measures that may be applied.
The application of AMS to the detection of 14 C makes possible a new class of sensitive experiments in molecular biology. Such experiments inherently produce large numbers of samples for the determination of biological variability in molecular interactions. The samples vary in 14 C concentration over many orders of magnitude. We added TiH 2 to aid the reduction of CO 2 by zinc in a sealed tube to reproducibly make graphite without sample cross-contamination. The CO 2 is transferred from a combustion tube to the reaction tube through a disposable plastic manifold. The sealed tubes are heated to a single-reaction temperature in a muffle furnace. The process is complete within 5 h. Bulk isotopic fractionation in the finished graphite is less than 0.5%.
We have studied DNA adduction with 14C-labeled nicotine and nicotine-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by accelerator mass spectrometry (AMS) in mouse liver at doses equivalent to low-level exposure of humans. The dose ranges of nicotine and NNK administered were from 0.4 μg to 4.0×102 μg kg b.w.-1, and from 0.1 μg to 2.0×104 μg kg b.w.-1, respectively. In the exposure of mice to either nicotine or NNK, the number of DNA adducts increased linearly with increasing dose. The detection limit of DNA adducts was 1 adduct per 1011 nucleotide molecules. This limit is 1-4 orders of magnitude lower than that of other techniques used for quantification of DNA adducts. The results of our animal experiments enabled us to speculate that nicotine is a potential carcinogen. According to the procedure for 14C-labeled-NNK synthesis, we discuss the ultimate chemical speciation of NNK bound to DNA. From the animal tests we derived a directly perceivable relation between tobacco consumption and DNA adduction as the carcinogenic risk assessment.
The metabolism of three triazine herbicides (atrazine, simazine, and terbutryn) was studied in rat (Sprague-Dawley and Fischer strains), mouse, goat, sheep, pig, rabbit, and chicken by using in vitro hepatic 10000g supernatant or microsomal systems. Principal phase I metabolites were 4- or 6-monodealkylated-s-triazines; several observations, including studies with purified enzymes, demonstrated that phase I reactions were cytochrome P-450 mediated. There were species-related variations in rates of metabolism and in ratios of primary metabolites, although no strain- or sex-related differences were noted. Phase II products were glutathione conjugates of the parent compound and of the two monodealkylated products. Experiments with Fenton's reagent, which generates hydroxyl radicals, gave dealkylated 2-chloro-s-triazine, supporting the possible role of active oxygen radicals in the cytochrome P-450 mediated reactions.
AMS directly counts radioisotopes without interference from molecular isobars. No chemical or physical information other than a bulk isotope ratio is available from the usual AMS instrument. Chemical or biological significance of the isotope ratio depends on the definition of the sample prior to conversion to material used in the AMS ion source. We use AMS as a detector for microbore and plate-based separation techniques in quantifying the binding of 14C-labeled compounds to specific DNA and protein fragments. We discuss our methods of using these microbore and plate separations of biomolecules while controlling contamination from 14C in laboratory equipment and give examples.
Organic compounds labelled with 14C are routinely and widely used in clinical medicine and biomedical research. There are, however, considerable uncertainties in the current estimates of the absorbed doses to the body from 14C-labelled radiopharmaceuticals. In this paper a project is described using accelerator mass spectrometry (AMS) to perform investigations of the long-term biokinetics of 14C from various 14C-labelled pharmaceuticals in man. The use of the AMS technique makes it possible to follow the turnover of the radionuclide administered for extensive periods and also to decrease the activity administered.
The AMS facility at Lawrence Livermore National Laboratory (LLNL) routinely measures the isotopes <sup>3</sup>H, <sup>7</sup>Be, <sup>10</sup>Be, <sup>14</sup>C, <sup>26</sup>Al, <sup>36</sup>Cl, <sup>41</sup>Ca, and <sup>129</sup>I. During the past two years, over 30000 research samples have been measured. Of these samples, approximately 30% were for <sup>14</sup>C bioscience tracer studies, 45% were <sup>14</sup>C samples for archaeology and the geosciences, and the other isotopes constitute the remaining 25%. During the past two years at LLNL, a significant amount of work has gone into the development of the Projectile X-ray AMS (PXAMS) technique. PXAMS uses induced characteristic X-rays to discriminate against competing atomic isobars. PXAMS has been most fully developed for <sup>63</sup>Ni but shows promise for the measurement of several other long lived isotopes. During the past year LLNL has also conducted an <sup>129</sup>I interlaboratory comparison exercise.
Recent hardware changes at the LLNL AMS facility include the installation and testing of a new thermal emission ion source, a new multi-anode gas ionization detector for general AMS use, re-alignment of the vacuum tank of the first of the two magnets that make up the high energy spectrometer, and a new cryo-vacuum system for the AMS ion source. In addition, we have begun design studies and carried out tests for a new high-resolution injector and a new beamline for heavy element AMS.
The occurrence and distribution of selected pesticides and their metabolites were investigated through the collection of 837 water-quality samples from 303 wells across the Midwest. Results of this study showed that five of the six most frequently detected compounds were pesticide metabolites. Thus, it was common for a metabolite to be found more frequently in groundwater than its parent compound. The metabolite alachlor ethanesulfonic acid (alachlor-ESA; 2-[(2,6-diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfonic acid) was detected almost 10 times as frequently and at much higher concentrations than its parent compound alachlor (2-chloro-2‘,6‘-diethyl-N-(methoxymethyl)acetamide). The median detectable atrazine (2-chloro-4-ethylamino-6- isopropylamino-s-triazine) concentration was almost half that of atrazine residue (atrazine plus the two atrazine metabolites analyzed). Cyanazine amide [2-chloro-4-(1-carbamoyl-1-methylethylamino)-6-ethylamino-s-triazine] was detected almost twice as frequently as cyanazine (2-chloro-4-ethylamino-6-methylpropionitrileamino-s-triazine). Results show that information on pesticide metabolites is necessary to understand the environmental fate of pesticides. Consequently, if pesticide metabolites are not quantified, the effects of chemical use on groundwater quality would be substantially underestimated. Thus, continued research is needed to identify major degradation pathways for all pesticides and to develop analytical methods to determine their concentrations in water and other environmental media.
Atrazine is an extensively used herbicide in the USA. Our objective was to determine the absorption and metabolism (detoxification) of atrazine in human skin. Percutaneous absorption of atrazine in human skin from four sources was examined utilizing a flow-through in-vitro diffusion system. About 16.4% of the applied dose was absorbed by the skin. Radioactivity in the receptor fluid at 20 h was less than 5% of the administered dose. The highest concentration of the applied dose was found in the skin supernates, where 12.0% of the dose (68 nmol) was recovered. Some metabolites of atrazine were identified by thin layer and high pressure liquid chromatography after extraction of receptor fluid and the skin supernates. Two metabolites of atrazine [2-chloro-4-ethylamino-6-amino-striazine (desisopropylatrazine) and 2-chloro-4,6-diamino-s-triazine] were found in the receptor fluid and the skin supernates. An additional metabolite (2-chloro-4-amino-6-isopropylamino-s-triazine) was found in the skin supernates. Since desisopropylatrazine represented about 50% of the total metabolites formed during percutaneous absorption, cleavage of the N-isopropyl to the amino product was a key step in the metabolism of atrazine. Further metabolism may proceed by cleavage of the N-deethyl group to give totally dealkylated atrazine. The biotransformation of atrazine was studied in skin microsomal fraction supplemented with an NADPH-generating system. In analogy to metabolism during percutaneous absorption, atrazine was metabolized to its deisopropyl and deethylpropyl derivatives. In addition, 2-hydroxy derivatives of atrazine were formed by the skin microsomal fractions. The biotransformation of atrazine by skin microsomal enzymes indicates the metabolic capacity of the tissue. Cutaneous metabolism of atrazine may be an additional route by which human skin detoxifies the pesticide following topical exposure.
Ultrasensitive SIMS with accelerator based spectrometers has recently begun to be applied to biomedical problems. Certain very long-lived radioisotopes of very low natural abundances can be used to trace metabolism at environmental dose levels (⩾ z mol in mg samples). 14C in particular can be employed to label a myriad of compounds. Competing technologies typically require super environmental doses that can perturb the system under investigation, followed by uncertain extrapolation to the low dose regime. 41Ca and 26Al are also used as elemental tracers. Given the sensitivity of the accelerator method, care must be taken to avoid contamination of the mass spectrometer and the apparatus employed in prior sample handling including chemical separation.This infant field comprises the efforts of a dozen accelerator laboratories. The Center for Accelerator Mass Spectrometry has been particularly active. In addition to collaborating with groups further afield, we are researching the kinematics and binding of genotoxins in-house, and we support innovative uses of our capability in the disciplines of chemistry, pharmacology, nutrition and physiology within the University of California. The field can be expected to grow further given the numerous potential applications and the efforts of several groups and companies to integrate more the accelerator technology into biomedical research programs; the development of miniaturized accelerator systems and ion sources capable of interfacing to conventional HPLC and GMC, etc. apparatus for complementary chemical analysis is anticipated for biomedical laboratories.
The excretion and metabolism of atrazine and procyazine were studied in pigs. Pigs were used because of their close modeling of human metabolism. Young (3-5 months) Pittman-Moore miniature pigs were dosed under anesthesia via a stomach tube with 0.1 g of the commercial grade herbicide dissolved in ethanol. Urine samples were collected from a drain in the floor of the metabolic cages. The urine samples were extracted and subsequently analyzed by gas chromatograph using the Hall electrolytic conductivity detector in the nitrogen mode. A column clean-up procedure using deactivated alumina was employed to further purify the urine extracts for analysis using electron-capture or mass spectrometric detectors.Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] and its metabolites were detected in the urine for slightly more than 24 h. Procyazine (2-[(4-chloro-6-cyclopropylamino)-s-triazin-2-yl)-amino]-2-methylpropionitrile) and its metabolites were not detected in the urine after about 10 h. Gas chromatographic/mass spectrometric analysis of pig urine extracts showed conclusively that the parent herbicide was being excreted. In addition, deethylatrazine [2-chloro-4-(amino)-6-(isopropylamino)-s-triazine] was identified as an atrazine metabolite, and several other compounds were identified as impurities in the dosing samples. If the findings of this research are extrapolated to monitoring studies in man, s-triazine herbicide may be expected in the urine within 24 h of exposure and primarily as the parent compound.
Analytical methodology for the separation and characterization of s-triazine herbicide residues in urine was developed. In the sample preparation procedure developed, a urine sample at pH 12 was extracted with hexane three times, using sodium chloride as an emulsion inhibitor. The combined hexane extract was dried by passing it through a sodium sulfate column and concentrated by rotary evaporation. The sample was transferred to a graduated centrifuge tube and further concentrated to 0.5 mL under a stream of dry nitrogen. The sample was analyzed by gas chromatography using the Hall electrolytic conductivity detector (HECD) in the nitrogen-specific mode. The method is advantageous in that it is simple and fast, lending itself to use as a routine method by technical personnel.
Studies on metabolism and excretion of atrazine in man are not available in the literature. The present study has investigated human exposure to atrazine during its industrial production by means of assessment of ambient exposure and determination of free atrazine in urine. Four workers exposed to atrazine during its manufacture and packaging in a production plant, volunteered for the study. Atrazine was determined in airborne dust of the working environment obtained by personal sampling, on skin pads according to the WHO standard method, and on the skin of the hands of the workers by means of a washing procedure. Urine was collected before, during, and after exposure. A 24 hr collection before the first workshift, all the urine voided during the monitoring period, subdivided in 8 hr fractions; and one or more 12 hr samples after the end of the exposure period were collected.
Rats were given atrazine (2-chloro-4-ethylamino-6-(isopropylamino)-s-triazine) in drinking water for 1 or 3 weeks at 0.1 (0.45 mM), 0.2 (0.9 mM) or 0.5 g/l (2.3 mM) concentrations of the commercial agent. They excreted at both time points as the principal metabolite 2-chloro-4-ethylamino-6-amino-s-trazine in a dose-dependent fashion. The same urine test was applied on 6 railway men engaged in the weeding operation of railway lines with known atrazine exposure measured with hygienic techniques in their breathing zone. The spectrum of their atrazine metabolites was comprised of fully N-dealkylated atrazine and 2-chloro-4-ethylamino-6-amino-s-triazine. The sum of the two urinary metabolites reflected quantitatively the exposure.
The genetic toxicity of atrazine, a member of the s-triazine herbicides, was reviewed with the objective of classifying the chemical. Atrazine has been subjected to a broad range of genetic tests with predominantly negative results. Some publications, specifically those measuring dominant lethality in mice and bone marrow clastogenicity in rodents, reported conflicting results across two or more independent tests. Two approaches were employed to evaluate and interpret the results. The first approach attempts to classify each type of genetic endpoint as positive or negative and resolve test conflicts by critical assessment of the study and detailed data. This is the more traditional "expert judgement" approach to hazard assessment. The second approach employs a computer-assisted weight-of-evidence method of data analysis. This approach does not require resolution of conflicts but uses all data sets to arrive at a classification of hazard. The first approach was able to resolve some conflicts but not all. Use of the "expert judgement" results in an equivocal conclusion and classification. Use of the weight-of-evidence method resulted in a conclusion that atrazine does not pose a mutagenic hazard. The weight-of-evidence scheme is proposed to be a more practical and relevant approach for assessing complex data sets.
2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a heterocyclic amine mutagen found in cooked meats and is carcinogenic in mice and rats at high doses (mg/kg body wt). Humans, however, are exposed to low amounts (p.p.b.) in the diet, and the effects caused by exposure to human equivalent doses of MeIQx have been difficult to determine accurately. We report on the effect of MeIQx exposure on liver bioavailability, hepatic DNA binding and MeIQx persistence in both liver tissue and liver DNA after acute (24 h), and subchronic (7 day and 42 day) exposures in male Sprague-Dawley rats. Male Sprague-Dawley rats were administered [2-14C]MeIQx either by gavage or in the diet for 1, 7 or 42 days (1 x 10(-6) mg/kg day up to 3.4 x 10(-2) mg/kg day dose) and the [2-14C]MeIQx was measured by accelerator mass spectrometry (AMS). Assessment of the kinetics of hepatic MeIQx DNA adduct formation over 42 days (1.1 x 10(-4) mg [2-14C]MeIQx kg daily dose) shows that steady-state [2-14C]MeIQx tissue concentrations of 138 +/- 15 pg/g liver and DNA adduct levels of 113 +/- 10 ag adduct/micrograms DNA were reached at 14-28 days and 28 days respectively. The relationship between administered dose and either hepatic MeIQx DNA adduct levels or MeIQx tissue levels are linear for the 24 h, 7 day and 42 day exposures. Furthermore, MeIQx adducts persist for at least 14 days after exposure ceases. These data suggest that bioavailability and DNA adduction by MeIQx increase linearly with increasing dose for both acute and subchronic exposures. These data also show that MeIQx DNA adducts are useful in predicting daily exposure and support a linear extrapolation in the risk assessment of MeIQx. However, the quantitative relationship between DNA adducts and tumor formation will also depend on the specific tissue and the subsequent steps needed for tumor progression.
Atrazine exposure was evaluated in six manufacturing workers by personal and biological monitoring. Total atrazine exposure varied from 10 to 700 mumol per workshift and total urinary atrazine excretion accounted for 1-2% of the external dose. The spectrum of the urinary atrazine metabolites comprises bi-dealkylated (80%), deisopropylated (10%), deethylated (8%) and unmodified atrazine (2%). The metabolites are eliminated in urine in slightly longer than 24 h: 50% of the amount is excreted in the first 8 h following the workshift.
Enzyme-linked immunosorbent assays (ELISAs) are reported for the detection of atrazine and its principle metabolite in human urine. The ELISAs can be used with crude urine or following extraction and partial purification by methods described in this report. GC, MS, and HPLC techniques were used to confirm and complement the ELISA methods for qualitative and quantitative detection of urinary metabolites. A series of samples from workers applying this herbicide confirmed a mercapturic acid conjugate of atrazine as a major urinary metabolite. The mercapturate was found in concentrations at least 10 times that of any of the N-dealkylated products or the parent compound. Atrazine mercapturic acid was isolated from urine using affinity extraction based upon a polyclonal antibody for hydroxy-s-triazines and yielded products sufficiently pure for structure confirmation by MS/MS. In a pilot study monitoring applicators, a relationship between cumulative dermal and inhalation exposure and total amount of atrazine equivalents excreted over a 10-day period was observed. On the basis of these data, we propose that an ELISA for the mercapturate of atrazine could be developed as a useful marker of exposure.
Triazine herbicides, used extensively in the United States, have not been assessed adequately for carcinogenicity in humans. This study evaluated the mortality experience during the period 1960-1986 of 2,683 men with definite or probable manufacturing exposure to triazine herbicides and 2,234 men with possible exposure to triazines. Standardized mortality ratios (SMRs) were computed as the observed numbers of deaths among study subjects divided by the numbers expected on the basis of general population mortality rates. Subjects with definite or probable exposure to triazines had a favorable mortality experience, compared with U.S. men [all causes, SMR = 72, 95% confidence interval (CI) = 58-89; all cancer, SMR = 85, CI = 46-142]. This group had an increase in deaths from non-Hodgkin's lymphoma (NHL) (3 observed/0.78 expected, SMR = 385, CI = 79-1,124). Two of the decedents with NHL had worked for less than 1 year in triazine-related jobs. Among the 2,234 subjects with possible triazine-related work, the mortality rate for all causes combined was similar to the rates of U.S. men. There was only one confirmed death from NHL. On balance, the results were consistent with previous investigations, which have found no convincing evidence of a causal link between triazines and cancer. However, the exposed cohorts were relatively young and had, on average, only 18 years of follow-up. In particular, results were imprecise for subjects having both a long duration of exposure and long potential induction periods.
Compounds of the s-triazine family are among the most heavily used herbicides over the last 30 years. Some of these derivatives are suspected to be carcinogens. In this study the identity of specific phase-I enzymes involved in the metabolism of s-triazine derivatives (atrazine, terbuthylazine, ametryne, and terbutryne) by human liver microsomes was determined. Kinetic studies demonstrated biphasic kinetics for all pathways examined (S-oxidation, N-dealkylation, and side-chain C-oxidation). Low K(m) values were in a range of about 1-20 microM, whereas high K(m) values were up to 2 orders of magnitude higher. For a correlation study, 30 human liver microsomal preparations were screened for seven specific P450 activities, and these were compared to activities for the metabolites derived from these s-triazines. A highly significant correlation in the high-affinity concentration range was seen with cytochrome P450 1A2 activities. Chemical inhibition was most effective with alpha-naphthoflavone and furafylline at low s-triazine concentrations and additionally with ketoconazole and gestodene at high substrate concentrations. Studies with 10 heterologously expressed P450 forms demonstrated that several P450 enzymes are capable of oxidizing these s-triazines, with different affinities and regioselectivities. P450 1A2 was confirmed to be the low-K(m) P450 enzyme involved in the metabolism of these s-triazines. A potential participation of flavin-containing monooxygenases (FMOs) in sulfoxidation reactions of the thiomethyl derivatives ametryne and terbutryne in human liver was also evaluated. Sulfoxide formation in human liver microsomes as a function of pH, heat, and chemical inhibition indicated no significant involvement of FMOs. Finally, purified recombinant FMO3, the major FMO in human liver, exhibited no significant activity (< 0.1 nmol (nmol of FMO3)-1 min-1) in the formation of the parent sulfoxides of ametryne and terbutryne. Therefore, P450 1A2 alone is likely to be responsible for the hepatic oxidative phase-I metabolism of the s-triazine derivatives in exposed humans.
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