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The UOEH Association of Health Sciences
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The UOEHAssociation ofHealth Sciences
J
UOEH28i.2.] ,157-171 (20061/･ 157
rOrigina]1
Inorganic
Effect on
Activity
Lead Exposure in Battery and Paint Factory:
Human Sperm Structure and Functional
N NAHAi･:' and A Roy CHowDHuRy2
i
Pq)aitment qfPh.vsioiog}/,
CoUege ofMedicat
Science,
Bharatpuny
2industrial R)xicolog.v Division, Regional
Occt{pationat
Health
ReseaTz'h,
Salt
Lake,
Kblkata
700 09f,
India.
Chinvan Distric4 iVepaL
Centre, indian Council (if Medical
Ahstract:
Key wonts:
Lead is
one of the industrially important heavy metals that causes male repro-
ductive
impairment among battery and paint
factory workers, but informatien on
the structure-function integidty
ofhuman spermatozoais still limited. Therefore,
it was necessary to investigate the effect of lead on sperm structure and func-
tional activity in these workers. Oligozoospermia with concomitant lowering of
sperm protein
and nucleic acid content and the percentage of sperm DNA
hyploidy
(P<O.OOI)
suggested the diminution of sperm cell production
after
occupational lead
exposurc. Low sperm vitality and hypoosmotic swelling
percentage
along with high
malondialdehyde contcnt and altered seminal
plasma
ascorbate level (P<O.OOI)
indicating damage of sperm cell surface,
might be due to high membrane lipid peroxidation
and failure of non-enzymatic
antioxidant protectjon
after Iead exposure. Alteration of sperm membrane sur-
face was also evidenced from scanning electron microscopy and further
authen-
ticated
by atomic and lateral
forcc
microscopy.
Lowering of sperm velocity,
gross
and forward progressive
motiljty with high stationary motiJe spermatozoa
(P<O.OOI)
suggested retarded sperm activity among the
exposed workers,
which was supported by high seminal plasma imctose level and reduced activity
of sperm MPase (P
<O,OO1).Increased incidence of teratozoospermia was also
associated with high bleod and semen lead level (PbB,
PbS) (P<O.OOI).
TheTefc)re,
the results suggested that lead not only affccts the sperm count, but
also damages the sperm structurc and membrane integrity, motility and t'unc-
tional activity amon.o the battery and paint
factory workers.
sperm morphology, sperm motility, sperm membrane lipid peroxidation,
bloodl
semen lead,
lcad exposed workers.
(Reccived
1 November 2005, accepted 1O Apri1
2006)
*
Correspondance:
l)fi
iVibedita Ndeha
(ftiv-639@.v.
ahoo,co,in)
The UOEH Association of Health Sciences
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The UOEHAssociation ofHealth Sciences
[58 N N.,xtIA
& A ROY CHOVL,DHURY
Introduction
The diminutien
of semen quality
due
to
occupational exposure to heavy metals is a ma-
jor
problem
in the world [1-31,
Lcad exposure and moderate lcad
absorption preduced
an
alteration in fertility with decreased production
in spermatozoa among battery factory
work-
ers,
probably
due to the direct toxic eflfbct of lead [4-6],
Reduction in
sperm velocity, for-
ward progressive
(FP)
motility, density and count, low antioxidant profile,
increased
incident
of sperm cell
abnormality and membrane iipid peroxidation
along with high PbB and PbS
level was also prevalent
after occupational exposure to lead [7,
8], The action of seminal
antioxidant in spermatozoal lipid peroxidation
was essential for
the maintenance of native
structure, so complete loss of antioxidant action caused spermatozoal denaturation
during
lipid
peroxidation
[9],
Though a positive
correlation of lead metal with the seminal plasma
of oligo-, astheno- and teratozoospermia group was reported by Kasperczyk
[1O],
the
inter-
ference of inorganic lead to the hypothalamic-pituitary-gonadal axis and sperm characteris-
tics
has
been
the
subject of great
controversy since lead might havc a secondary effect on the
endoerine axis [1
1 - 141. Moreover, infOrmation on the possib]e
impact
of lead
on the struc-
ture
and the function of human spermatozoa is stiil ]imited, Therefore,
it
was necessary to
investigate the eilfect of lead on human spermatozoa of the battery
and paint
factory
workers
in these aspects.
Subjects
and Methods
Chemicats
and teagents
For sperm cell staining, hematoxylin monohydrate, light green
SF and Eosin
were pro-
cured from BDH, England (Gurr
Certistain). For
morphological, biochemical
and metal
analysis, the fo11owing
chemicals were purchased
from E-Merck, Germany: glutaraldehyde,
fbrmaldehyde,
diphenyl amine, 2-4 dinitrophenyl hydrazine, disodium hydrogen
phosphate
dihydrate,
sodium hydrogen orthophosphate, sodium hydroxide pellets,
sodium chloride, so-
dium bicari)onate,
sodium citrate
dihydrate, sodium potassium
tartarate, sodium cacodylate
trihydrate,
zinc sulfate heptahydrate, copper sulfate pentahydrate,
anhydrous ferric chloride,
resorsinol, D (-)
fructose, ascorbate, trichloroacetic acid, acetic acid glacial,
nitric acid, hy-
drochloric
acid, su]furic acid and hydrogen
peroxide.
BSA fraction V was obtained from
Sigma <St
Loujs,
USA). Thiobarbituric acid, orcinol monohydrate, RNA from torula yeust
and DNA from herring sperm were purchased
from Himedia, India. Tris, DPX mountant,
thiourea, bromine ampulle, Folin Ciocalteau's reagent, rectified spirit and abso}ute alcohol
were procured
from Glaxo, India.
Stud.v
design and selection ofsubjects
The present
study was conducted in accordance with the Helsinki
Declaration
(1983).
Prior
to
the
study, ethjcal clearance was obtained from the Indian Council of Medical Re-
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EffecC
efLead on Human Sperm 159
seurch (ICMR),
Government of India. Fifty (n=50)
non-occupationally exposed control
subjects (group
I )
of active reproductive age (31-45
years)
[15]
were randomly selected af-
ter
a proper
medical check up by a physician.
Lead-exposed workers of the same age group
were sele¢ted from battery and paint
manufacturing factories
in Kolkata, India,
and were di-
vided into two groups,
depending on the duration of exposure; low exposed group (group
ll:n=30) with 7 to 1O years
exposure fbr8 hours/day, and high exposed group
(group
M:
n=50) with the same daily
duration
of exposure, but fbr
longer periods,
i.e. more than 1O to
15 years
of lead exposure in the factories. Using interview technique as a tool for data col-
lection,
detailed
information
of the subjects was recorded en a pre-designed
proforma
(ques-
tionnaire),
and the
consent forms
for voluntary donation of blood and semen samples were
signed by the subjects.
Coltection
ofbiological
samples
Semen samples were collected from the subjects in a clean, dry,
sterilized,
wide mouth,
well stopper gEass
vial by masturbation [15,
16].
O.5
ml semen was stored at "20 ℃ in
a
lead-free
storage vial for
lead
content analysis. 2 ml of venous blood was collected asepti-
caliy and 1 ml was stored at L 20 ℃ in a lead-free heparinized
vial for
metal analysis,
Analysis
ofsperm
ceun4 morpholog.v, motilio' and cetlular integrity
Sperm count, motility and morphology were deter'mined
[15].
Sperm velocity [171,
vi-
ability by dye exclusion supra vital staining technique Ll8J
and hypoosmotic swetling test
(HOST)
[19]
were also measured at 400 × and 1000 × magnifications (CH20i,
Olympus,
India).
The cell cycle phase
disnibution of sperm head DNA was studied by flow cytometer
(FACS
Vantage,
Beckton
Dickinson,
USA) [201,
and sperm cell surface was analyzed by
scanning electron microscopy (SEM)
at 10000 × and 15000X magnifications (JSM
5200,
Jeol Pvt.
Ltd,,
Japan) [2t]
and also by atomic and lateral force microscory (AF[M
and LFM)
at native condition using 75000 × and 80000
× magnifications (Nanoscope
E,
Digital
Instruments Inc., USA) [22],
where LFM image indicated surface roughness at native condition,
based on the surface composition as per
the maker's opinion (Digital
Instruments Inc., USA).
Biochemicat analysis qf'semen
After liqucfaction, semen samples were centrifuged at 800 ×g for
10 minutes, and sperm
was separated from the seminal plasma.
Sperm ArPase activity [23],
sperm membrane lipid
peroxidation
[24],
total sperm protein
[25]
and nucleic acid contents L26,
27], as well as the
amount of seminal plasma fructose
[28]
and ascorbate [29],
were determined by spectropho-
tometer (DU64,
Beckman Instrument Inc., USA).
PbB and PbS analvsis
"
Blood and semen samples of the subjects were acid digested
in
the
digestion
chamber of
Ethos D Microwave Labstation with Terminal 20 operating system (Milestone
Sr], Italy) and
The UOEH Association of Health Sciences
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160 N N,xHA & A ROy CHOWT)Hl]RY
then absorbance was taken at 283.3 nm usi"ng atomic absorption spcctrophotometer (GBC
AVANTA AAS. software version 1.33, GBC Scientific Equipment Pvt, Ltd,, Australia) at-
tached to a GF 3000 graphite
furnace [30].
StatisticaZanalysis
The data
obtained from
the
controls and exposed groups
were compared, and one-way
ANOVA, two-tail
tt'
test
and Scheffe's
`F'
test
were carried out for
level of significancc us-
ing the computcr based statistical software SPSS'/'"',
version 10.0 for
Windows (SPSS
Inc.,
USA).
Results
Analysis qfsuhiects
as per the questionnaire
data
The demographic details of the subjects is shown in [fablc 1. which indicates all the sub-
jects
belonged to lower socio-economic status and most of them were addicted to smoking
and consumption of alcohol as well as gutkha
and panparag,
There was no reproductive
disorder among the subjects, but few workers were going
for treatment, as they werc is-
sueless (Tab]e
1), Questionnaire
analysis also revealed that the majority of the subjects of
group
I
(18.7596),
ll (19.35%)
and M (16,12%)
were 35 years of age.
Table 1. Demographic details of zhe subjeets
Parameter Control
(group
1)
n=50 (%)
Exposed(.o,roup ll & M)
n-30 & 50
(
Uf6
)
Abstincncc
time
Married for 5 years-
One child
1["Jo
children
More than two children
[ssueless, undergoing treutment
Histury of miscarriage
Lower seeie-cconomic status*
Smoking bidi
for
1O
years#
Alcohol (country
]iquor> consumptiun for
10 yearsGt]tkha/punparag
consumed for 7 years#
Reproductivedisease/disordcrs
4days
100
15
50
35
Ni]
Nil
1OO
20
IS
10Nil
4days
1OO
25
40
30
5
Nil
100
80
95
50Nil
'':
Wii,es
not taken
any pill
and male counterpart never uscd any contraeeptive devi¢es
': Monthly income: aycragc Rs, 3000,OO (1
US $=Rs.
45.00 IC)
#: Contained
mainly tobaceo (hiddi),
lime and tobacco Cgutklia),
dry areca nut aiid catechu (panparag),
used wide]y in India
n=Sample size
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Effect
ofLead on Human SpeiTn 161
Eff}2ct
qftead
on spetim count, totat sperm piotein
and nucteic acids content
There
was a significant (P
<O.OO1)
reduction in
sperm count in
the
workers exposed to
lead fumes and dust in the factories when compared to the non-occupationally exposed con-
trol persons
(Table
2), The controls (group
I)were normozoospermic in nature, and the
high lead exposed workers (group
M) were oligozoospermic, but the low lead exposed
workers (group
ll )
werc in bctwccn
the
normozoospermic and oligozoospeTmic category
L15].
There was no sign of polyspcrmia
after lead exposure. In support of sperm count, to-
tal sperm protein
and nucleic acids contents were estimated, which showed a significant
(P<O.OOI)
decrease in the same comparable groups
(Ttible
2), The flow cytometrical
analysis of sperrn head nuclear DNA also showed a high percentage
of hyploidy (<n DNA)
at sub Gi phase
as the exposure increased ([[hble
2).
'1iable
2. Effect of ]ead on sperm ceunt, total sperm preteln
and nucleic acid eontents
Group(n) Sperm count
uoetml)
Sperrn protein
(pglmg
eclls)Speni]
RNA
{Ugfmgcclls)Sperm
DNA
(nglmgcclls)Sperm
DNA
hyploidy(Y6)
Control(I)
(50)Low
exposed(ll)
(30)High
exposed (M)
(50)
137.47+39.42
74.70+15.44ai
28.97± lo.62aib]
27,l7 ±4,91
14.61±2.gsat
5.5712.lgaibT
l922 ±4.42
7.01±1.37mi
2.04±O.67,,ibL
72.01±8.97
30,17±8,32ai
11.8±7.2
l7.9±1.soai
4.9813.44?i]bl
34.5]t,4.2o,,ihi
S': P<O.OOI (comparedwith
-group
I ).
bi: P<O.OOI (eompared
with group
II ),
n=sainple size.
Valucs givcn
wcre thc mcan ±SD of thc control and exposcd groups
EZIlact
qflead
on sperm morphotogical ahnormatit.v
Gross morphological abnormality of spennatozoa was significantly (P<O.OOI)
hjgher
in both thc cxposcd groups with respect to the controls (Fig.
1). The low exposed group
(44.54
±2.93) and the high exposed group
(60,04
±7,82) were teratozoosperrnic in naturc,
whereas the abnormality of the control group (33.75
±4.89) was within the normal range
[15].
The present
study also revealed that tota] sperm head, mid piece
and tail abnorrnalities
increased significantly (P
<O.001) after exposure (Fig.
1),
Sperm
cell membrane integriA. ;
c{fiter occmpational lead eJrposuiie
Spcrm viability and HOST pcrcentage
was significantly (P
<O.OOI) reduced in both the
exposed groups
and in between the two exposed groups
(Tbble
3). Lipid peroxidation
of
sperm tnembrane showed significant (P
<O.OOI) high malondialdehyde (MDA)
content in
group
ll and M workers in comparison to the control subjects of group
I,which indicated
loss of sperm membrane integrity after occupational lead exposure (Table
3), This finding
was supported by a significant (P
<O.OOI) low level of semina]. plasma
total ascorbate with
concomitant high value of dehydroascorbate (DHAA)
concentration in sentinal plasma
of
the same comparab]e groups
(Tlablc
3).
The UOEH Association of Health Sciences
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The UOEHAssociation ofHealthSciences
162 N NAHi!
& A ROY CHOWDHURY
(?o
Table 3.
go70
60
5040
3020
10o
Fig. 1.
Effkicts
of
plasma
*
Gross Tota]
hcad Tota]
mid Tota]
tail
abnotinulity abnormality piccc abnermality
abnornialitv
Variations
of human sperm morphologicaL abnorrnality after ]ead
exposure.
V}ilues
given
were the mean ±SD of the contro] and exposed .oroups.
where asterisks indicated significant difTerence of the cxposcd groups
fi'om
the correspondin...o control grnup
('P
< O,OOI),
ua:Group I, -iGroup ll, EIew/Group M.
Iead on gperm viability, membrane lipid peroxidation
and antioxidant profile
of serninal
Group(n) Spennviabi]ity
staining(96)Sperm
viabi]ity
by HosT (96
)
Spcrm mcmbrane
lipidperoxidation
(n
mole MDA 1
10S ceUs)
Seminal Setnina]
plasma total plasma
ascorbatc DHAA
(pglml)
(pghnl)
Control(I)
(50)I.ow
exposed (
ll )
(30)High
exposed (
llI
)
{50)
79.72± 6.01
60.22± 5.03tii
26.g3±'to.33nibi
63.40
±8.17
38.33±5.6o:i
1
9.35
.. 7.2ga,bi
24.53
± 7.62
73.80 ±ll.74"]
IS5.05
±24.22uibi
74.80 ±10.49
22.60±9,24
46.30± 5.95Hi
42,30±6.18"i
28.10
± 5.78uibi
70.10±6.s7uibi
ai: P<O.OO1 (compared
with gToup I ),
b":
P<O.OO[ (compared
with group
Vtalues given
were the mean ±SD of the control and exposed groups
II ),
n :z'='
sample size,
SEM, AFM and LFM further authenticated the alteration of sperm surface morphology
afier occupational lead exposure, where 20 sperm cells per
sample (n=
15 samples in each
group that showed high MDA, low sperm viability and altered seminal plasma
ascorbate
level) were examined, SEM exhibited a sharp depression and granular
texture on the sperm
cell surface of the exposed workers compared to
the smooth membrane surface of the
con-
trol
persons
(Fig,
2),
hence confirming the
previous
membrane integrity
study of the sperma-
tozoa after lead exposure ([[hble
3). AFM (Fig.
3) and LFM (Fig.
4) images of sperm head
The UOEH Association of Health Sciences
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The UOEHAssociation ofHealthSciences
EfTlect
oI'
Lead on Human Sperm 163
surface supported the SEM
deterioration of sperrn membobscrvation
at native condition and thus finally
ranc structure after occupational exposure to lead.confirmed
the
a:
Control (
× 15000),
b:
Low exposcd (
× 1OOOO).
e: Low exposed (
×
1
5000).
Fig. 2.Reprcsentative SEM imag,e of control and ledd exposed human spermatozoa.
a) ConTro] hurnan sperm (group
T) showed normal oval shaped head (H),
mid piecc
(M)
and proximal
part
or tail (T),
Well defined acrosomc area
(A)
and smooth membrane surface was prominent.
Thc uverage length
of sperm hcad was 3.5 pm and average brcadth
was 2.S
y.m.
Mid piccc
was approximately 1.7 pm long and 1.2
pm wide (× 15000).
b) Exposed human spcrm (group
II)showcd
depression
(.)on
swcll mid
piecc
(M),
Granularity
(n)
appeared on spenn hcad (H)
and mid piece
surfaee. (A)
was thc
acrosome area and (T)
was the part
of the rail. The
averagc lcngth and breadth of spcrm head was 3.8
pm and 2,2
pvn,
rc-
spective]y< ×1OOOO).
c) Exposcd human sperm (group
M) showed abnormal elongt'tted swell mid
pieee
(M)
with depression (
"
).
CT)
was part
of tuil. Granuiar texture
(fi)was
noticed en amorphous shaped sperm hcad (H)
surface. The av-
erage length and breadth of sperm hcad was 2,9
p,m
and 2.2
FLm.
rcspcc-
Lively( ×15eOO),
The UOEH Association of Health Sciences
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TheUOEHAssociation ofHealth Sciences
164 NNAHA&ARoyCHOWDIIURY
a
g
×.. /-
t)S'
'....U/"'/ x o
sca
""tdW
P" z tao.aoo
n-xdiv
hsp.ooe
b.m.'
-
,Mi.lll/,;･#""'
t･
1'O
)sX.
Il
//
Z,e 'NL!
x o.sont ps/aiu
PH z 4oo,coD n-taiu
hspM.e05
Fig. 3.Representative three-dimensiella]
AFM imae.e
of cDntrel and Iead exposed humun sperm head surl'ace.
a) The brighter area (t)
of the ovai shaped contro] humaii sperm head
(greup
I) indicated
smputh
mcmbranesurface and the darkcr Crcddish-browll) areas represented the luwer pertion
of same cell (1)
or
the
depression
between two acljacent cells (2,3).
SEM could not reyeal this patteni
due Lo gold eoating on
thc
top of the
sump]e, thereforc
this
image was considered as the true stiueture of control sperm head
suTfacc at nativc condition (×80000).
h) Expf)s'ed
humEtn
spcrm head (group
ll) was charactcri7ed by unes,en tnembrane surfacc (
- )
ol' dark
(reddish-broN-'n)
colour, hence
thc surface smooth"ess of the exposed cell was not eomparable with that oi'
the contrel one cven at native condition C× 75000).
a
hsp.MS
t:V
'x
hspt2.0as
b
tii-iv
Fig.
4.Representative thrcc-dimensional LFM image of control and lead cxposed human sperm head surrace.
a) The centrol humati
spenn head
(group
1
) surfuee was characterized by hill-like patterm,
made by darkcr
(reddish-brown)
dcprcssions/pits {
L) rrange:
5C)-75nrn diaineter] and brighter elevationsfblebti like
structurcs (
n )
1range:
87.S - 1OO nm heiglitl. Higher
the
elevations, brighter
thc arca indicated
increased
surface roughness ("),
and lo",er the depressio]s, darkcr the area represen[ed compuTutively smootlier
$uiface C
l
)
of the
control speiin hcad
at native condition C
×
80000).
b)
The exposed human sperm head Cgroup
M) surface was also characterized by different types of depres-
siens/pits (
L
)
[range:
62-78 nm diarneter] and elevationstb]cbs (a)
rrange:
93-11O nm height].
Here
a]so the brighter
elevated areas indicated rough surface (
n).
and darker (rcddish-brown)
depressed areas
showed comparatively smoother zones (
L
)
at naLive conditien, which was difieren[
from that
ot'
the eon-
trol
image,
hence
difi'erent
surfacc morphology of sperm head might bc due to occupational exposure to
lead{×75C)OO),
The UOEH Association of Health Sciences
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The UOEHAssociation ofHealthSciences
Effect ef Lead on Human Sperm 165
Functionat activit.y of'spemialo4".oa
c4leroccapationat expost{re to lead
Sperm velocity, gross
and forwand and progressivc
(FP)
motility were significantly (P
<
O,OO1)
decreased
in lead exposed workers (group
ll
and M) of battery
and paint
factories
(Fig.
5). Significantly (P
<O.001) high level of seminal plasma
fu]ctose and reduced activ-
ity of sperm iffPase were also observed in the same comparable groups
after occupational
lead exposure (Fig,
5). Further, in the present
study, sperm velocity, gross
and FP motility
decreased proportionately,
whi.le SM showed an inverse relationship in respect to duration of
lead
exposure (Fig.
5). The decrement ratio of sperm velocity, gross
and FP motility in group
I, ll and M were approximatcly 15: 5: 1, 3: 2: 1 and 2.5: 1.8: 1, respectively, whereas the
increment ratio of SM spcrmatozoa of the same comparable groups
was approximateJy 1: 2:
3. This study also showed that the gross
sperm motility and FP of all the three groups
was
much lower than the respective reference values Ll5],
6
4
2
o
･-e-Spenn A'1'1'ase activity
C
,u Mole Pi liberated/mg protein)
*
*
4
3
2
1
o
-- Fructose (,mgtml)*
1OO
80
60
40
20
o
Group IGroup ll Group M
*
t"
*
tt
*
Z:s;:;k:S:Z:t:""):t:t:t:t-'tn.ws,:sss::::stttt.ttttttt.tttt):ttt:t:t:t:ISIi:il;i]i･tk:Sst:::::t:
tt"ttttttttt"ttttt
Ch'oup IGroup llGroup -
rk
lt .
Sperm velocit>, (ulsec.)
Gross motilit}, Cqi,)
FP {%) SM (`Ja)
Fig. 5. Effect of lead on sperm cell activity of control and exposed subjects.
Valucs giyen
wcrc thc mcan ±SD of thc control and exposed groups.
where asterjsks indicated significant difTeren¢e of the expesed
groups from thc corresponding control group.
*P<O.OOI.
E2Z:Ci'oup T, D;Group ", ma:GroupM,
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The UOEHAssociation ofHealthSciences
l66 N NAI{A & A ROY CHOWDHURY
PhB and PbS ievel
Lead concentration in whole blood and semen was increased significantly (P
<O,Oel) in
both the exposed groups
and also between the two exposed groups
([Ibble
4). The observed
value of PbB in both the exposed groups were higher than the WHO's pcrmissible
limit of
40 pg!dl.
[[hble
4.Body burden
of lcud
after
occupational cxpesure
Group(n) PbB (pg/dl) PbS (yg/dl)
Control(I)
C50)Low
exposed(n)
(30)High
exposed <M)
(50)
13,62±2,45
48,29±4,91-i
77,22 ±1,2s-ihi
3,99±1,36
10,85±O,75,,i
18,30±2.os"ihi
"]: P<O,OO] (compared
with group I ),
b': P <O.OO1 (compared
with group U ),
n=sampie size,
Va]ues given
were the mean ±SD of the control and
exposed groups
Discussion
The present
study was the very first
study in
lndia to deal with a working population
routinely exposed to lead fumes and dust in battery and paint
factories. The present
study
was undertaken with an idea to estal)lish a relation between occupational lead exposure and
structure function integrity of human spermatozoa among these factory workers, Several
earlier studies reported the direct toxic efifect of lead
on sperm characteristics, with an in-
creased incident of lead in several body fluids l3,
4, 31, 32]. Chronic lead exposure also
caused significant diminution of sperm count among workers [6,
14, 32]. The present
study
revealed a significant reduction of sperm protein
and NA content in the exposed factory
workers, which might be related to the diminution ef sperm cell production
after occupa-
tional lead
exposure. The whole event was manifested by oligozoospermia when compared
with the non-occupationally exposed control subjects of the same socio-economic status [5,
33]. Funher, the above finding was authenticated by fiow cytometrical cell cycle phase
analysis of sperm head DNA, where exposure-dependent decrease in intact DNA content
with consequent rise in hyploidy (<n DNA) at sub Gi phase
was evidenced among these
workers, suggesting lead-induced fragmentation of sperm head DNA [34-36].
The factory workers were exposed to lead dust, fumes and finer particles
that caused ad-
verse effects not oniy on sperm count, but also on sperrn morphology, depending on the du-
The UOEH Association of Health Sciences
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The UOEHAssociation of Health Sciences
Effcqt
of Lead on Human Sperm 167
ration and nature of exposure [2,
10, 13], Strikingly abnormal gross
sperm morphology
among the workers was evident in the present
study, which was supported by the
individual
abnormalities of total sperm head, mid piece
and tail after exposure, as revealed by light mi-
croscopy, and then authenticated by SEM, which further indicated
lead induced morphologi-
cal changes ofhuman spermatozoa after occupational exposure L37-39].
As cellular viability depends on the intact membrane structure [20,
40], dintinution of
sperm vita]ity and HOST percentage along with high lipid peroxidation
of sperm membrane
as well as corresponding non-enzymatic antioxidant (ascorbate)
profile
in
the
workers of
battery and paint
factories suggested the probablc
loss of sperm membrane integrity
after
ec-
cupational exposure to lead
[9,
41, 42]. This observation was supported by the surface
structure study of spermatozoa through SEM [38],
and then finally confirmed by AFM and
LFM at native condition [35,
36, 39]. Several earIier studies provided
information regard-
ing sperm merphology and ultrastructural damages, but till
date,
intemet
searches revealed
no such reports on surface analysis of spermatozoa aftcr occupational exposure to lead,
hence
the present
study reports the first
ever SEM, AFM and LFM image of lead exposed
human sperm cells.
Fructose was the main energy source for spermatozoal motility through fructolysis,
where membrane beund MPase plays
an active role required for the deve]opment
of pro-
gressive
fbrward
flagellar
motion L5,
43,
44].
Sperm velocity was the avcrage vclocity of al1
spermatozoa in one sample [15],
therefore related with gross
and graded
motile activity (FR
SM etcL.)
of spermatozoa. In the present
study, lowering of sperm velocity and gross
and FP
motility with concomitant rise of SM were prevalent
alnong the lead-exposed factory work-
crs, which suggested that retarded sperm activity might be due to lead-induced aiteration
of
normal fructolysis.
High seminal plasma
fructose
level
with consequent lowering of sperm
MiPase activity further
supported the above statement, and was also corroborated by several
previous
obseryations L7,
8, 35, 38]. Thus, the probable
explanation is: in normal physi-
ological conditions, fructose is utilized by MPase durillg imctolysis, but lead inhibited this
cnzyme by replacing Na', hence fructose was not uti]ized, rather it might be accumulated in
the seminal plasma of the affected factory workers [45,
46]. Further, the
association of low
sperm motiljty with low antioxidant level and concomitant rise in the rate of MDA produc-
tion in
both
the exposcd groups
of the present
study suggested the susceptibility of sperma-
tozoa to lipid
peroxidation
after occupational lead exposure [47,
48J. Also, a definite role of
lead in impaired sperm motility and poor
sperm DNA integrity was established in the
pre-
sent study [34-36].
In
the
present
study, deterioration
of sperm count, normal morphology, viability, mem-
brane
integrity
and motile activity wcrc associated with high PbB and PbS level in the work-
ers in
respect ofduration of exposure L1,
1O, 49], Moreover, moderate lead exposure caused
a reduction in sperm characteristi'cs among the factory workers [12],
and semen lead was the
indicator of the jndustrial exposure [50].
The UOEH Association of Health Sciences
NII-Electronic Library Service
The UOEHAssociation ofHealth Sciences
168 N NAIIA
&A ROY CHOWDHURY
Therefore,
in
conclusion it can be hypothcsized that lead exposure might be responsiblc
for high blood and semen lead levels in the battery and paint
factory workcrs, and the pres-
ence of lead in both these biological fluids indicated that lead might ¢ross the blood-testis-
barrier
and subscqucntly produce
detrimental effects on human spermatozoa ()f
the working
population
L5,
45, 511. The normal cell membrane is the pre-requisite
for the proper
func-
tion of the ccll, therefore
it
can be
pointed
out that
a subtle membrane defect in morphologi-
cally abnormal spcrmatozoa due to lead-induced lipid peroxidation
of
the membrane in
asso-
ciation with low antioxidant protection
might be responsible for impaired functional activity
of spermatozoa, i.e,
impaired
motility among these lead-exposed battery and paint
factory
workers in
the present
study. Smoking,
consumption of alcohol, etc. might be the probable
confounders in lead toxicity [52,
53], while the age of the subjects did
not show any correla-
tion after exposure, as active reproductive agc group
subjects were consideTed throughout
the study [15,
16, 54],
Acknowledgements
This
work was supportcd by a DST Grant
from West Bengal. ROHC (ICMR),
USIC
(Jadavpur
Univcrsity) and IUC (Indore
University) provided
the laboratory
facilities. Ac-
knowledgement
is due to Dr. B Manna, AD, NICED (ICMR),
Kolkata for statistical analysis
and Mr.
PK Debnath for
providing
a computer facility, Thanks are due to the sample do-
nors of the study without whose cooperation thc study would have
been incomplete.
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Effcct of Lcad on Human Sperm 171
52．Gomathi C，
Balasubramanium K，
Bhanu
NV ，
Srikanth V＆Govinda画ulu P（
1993）
：Effect of chrollic
alcoholism
on semen −studies on 且ipid
profiles
． Int J Androl
l6：175 …181
53，Practice ceminittee of the American Society for Reproductive Medicine （
2004）
：Smoking 乱nd inl℃質il
−
ity．
Fertil Steril
S1：ll81 −ll86
54．Check JH ，
Shanis
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Bollendorf
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バッテリーと塗料：ll
場に お ける無機鉛曝露 ヒトの精子の形態と機能 に対する影響
エヌ・ナハ112
，エイ・アール・
チャウドハリー2
］医科学大学 生理学教室 チトワ ン 区 ネパール
L
’
インド医学研究評議会 地域職業研．
究センター 産業中毒部門 カルカッタ インド
要 旨 ’
キーワード
鉛は工業的に重要な重金属の一つで，
バッテ リ ーや塗料⊥場労働者の男子に生殖障害
を引き起 こすことが 知 ら れ ているが，
ヒトの精 子 の形 態 と機能に対する影響に関 す る
情 報 は限られて いる．
従って，これ らの労働者に お ける精 了
の形 態 と機 能 に対 す る鉛の
影響 を研究することは重要である．
精 了の蛋 自と核酸濃度の低下を伴 う乏精液症 と精
子DNA の高倍数性の割 合 か ら，
職 業 性 の鉛曝露 によって，
精 了細 胞 の産生が低下する
事が示峻され る ．
精rの活動 性 の低．．
ド
，
高濃度のマロンディア ルデヒドと精 子細 胞 表 面
の障害 を示唆する精液アスコルビン酸レベルの変 化 を伴 う低浸透圧 ド
での膨 化 した精
子の割 合の増加 は鉛曝露 による膜脂質の過酸化 のE昇と非酵素的抗酸 化防御の低ドに
よる ものと考えられる，精子膜表面の変 化 は ま た ，
走査電子顕微 鏡によ り明 ら か にさ れ ，
原 子 問 力 顕 微 鏡 によりさらに確実にされた．
精液粘度の低ドと運動 能力を失った精子
を含む各方向 と前方への運動性の低下か ら ，鉛曝露 を受けた労働者での精子の活動性
の遅延が示唆 される．
このことは精液のフルクトースレベルの上昇と精子 のATPase の
活 性 の低下 に よりさ らに支 持 さ れ る．奇形 精 r・
の割合の増加 もまた，
血液 と精液中の鉛
濃 度 レベル の 上昇と鬨 連 がある．
従って，こ れ らの結 果 からバッテリーや 塗 料 T一
場労働
者で は 鉛は精壬数に影響 を与える の み で な く，精子の形態，膜の構 造 ，運 動 や機 能にも
障害 を与えることが示唆される．
精子の形態，精 子 の運動性 ，精子膜脂 質の過酸化，血液と精液中の鉛濃 度，鉛曝露労
働 者．
」∪OEH 〔産業医大誌）28 （
2）：157−171 （
20〔〕6）
N工工一
Eleotronio
Library
