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Reproductive hazards of lead exposure among urban egyptian men
Reproductive Toxicology, Vol. 10, No.2, pp. 145-151, 1996 Copyright © 1996 Elsevier Science Inc. Printed in the USA. All rights reserved 0890-6238/96 $15.00 + .00
0890-6238(95)02057-8
REPRODUCTIVE HAZARDS OF LEAD EXPOSURE AMONG URBAN EGYPTIAN MEN EMAN
A. EL-ZOHAIRY,* ASHRAF F. YOUSSEF,* SAID M. ABUL-NASR,*
IBRAHIM M. FAHMyt DAWLAT SALEM,:j: AZIZA K. KAHIL,* and MAHMOUD K. MADKOUR§ Departments of *Forensic Medicine and Toxicology, :j:Biochemistry, §Pharmacology, and tVenereal Diseases, Cairo University, Cairo, Egypt Abstract - Fifty-five urban Egyptian males, aged 20-40, were assigned to two main groups to study the effects of their exposure to lead (Pb). Group I, infertile men (INF, n = 30), was divided into environmentally exposed (INF-E, n 15) and environmentally and occupationally exposed (INF-EO, n 15). A matching group (II) of fertile men (F, n = 25) was divided into fertile, environmentally exposed (F-E, n = 10), which was the control group, and fertile, environmentally and occupationally exposed (F-EO, n = 15). Semen parameters (i.e., count, morphology, motility, and volume), blood and semen Pb levels, and reproductive hormonal indices (i.e., serum testosterone, FSH, and LH) were measured in all subjects. Lead levels were always higher in blood than semen. Semen lead levels were significantly higher in all groups vs. the control (F-E) group. While no changes were observed in testosterone levels across groups, variable effects on LH and FSH levels were observed. Infertile-EO subjects showed a definite pattern of impaired semen parameters in comparison with infertile-E. No abnormalities were detected in hematologic, hepatic or renal function.
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Key Words: lead; infertility; males; semen parameters; gonadotropins; occupational; environmental.
INTRODUCTION
include cement, chemicals, fertilizers, iron, steel, nonferrous metallurgy (lead and zinc), asbestos, and electric power, in addition to other minor sources of air pollution (e.g., printing and smelting). Furthermore, the lead constituent of dustfall over southern Cairo has reached 1,652 f.Lg/g of dust (40 to 1652 range) and its solubility has been estimated to be at least 23 to 53%, which increases its bioavailability in comparison with the lead in other countries (e.g., 10 to 20% in the UK) due to differences in atmospheric constituents and conditions (6). A significant correlation between a decrease in lead levels in gasoline and a decrease in blood lead levels has been shown (7). In East Germany, a highly significant (P < 0.0002) decrease in lead levels in blood and semen over 2 years was associated with a decrease in the lead level in gasoline (8). In Cairo, the blood lead level among traffic policemen has been estimated at 39.16 ± 2.12, the level for drivers at 34.61 ± 2.82, and the level for gas station mechanics at 36.6 ± 3.28 f.Lg/dL (9). El Samra and colleagues (10) measured mean blood lead levels among housewives, female rural employees, male rural employees, drivers, and policemen to be 11.2, 16.9, 17.5, 28.2, and 27.3 f.Lg/dL, respectively. Nasralla (3) estimated blood lead levels in traffic policemen to be as high as 39 f.Lg/dL and 62.7 f.Lg/dL in moderately and heavily exposed groups, respectively. It is worth mentioning that the threshold limit value (TLV) in Egypt is 0.20 mg/m 3 , which is close to that of the U.S. (0.15
Cairo, Egypt is considered one of the most polluted cities in the world (1,2). The lead concentration in the atmosphere of Cairo was measured at 10 f.Lg/m 3 in 1980 and 4.5 f.Lg/m 3 in 1984 (3), both of which exceed World Health Organization (WHO) guidelines of 0.5 to 1 f.Lg/m 3 (1,2). The U.S. Environmental Protection Agency (EPA) (4) has estimated that globally approximately 90% of the lead entering the atmosphere comes from the combustion of leaded gasoline. In Cairo, leaded gasoline has an average lead of 0.2 to 0.4 gIL added as tetra-ethyl lead, and is used without proper controls on car exhaust quality. Like leaded gasoline, leaded paints, which have inorganic lead compounds of more than 40% by weight, are also still in common use. Furthermore, there is an outdated network of lead pipes in use for the water supply, and the lead level officially allowed in Egyptian water is 0.1 mglL, which is double the level recommended by the WHO (5). Other topographic and environmental conditions contribute to high lead levels in the Cairo environment. For instance, industry activities in the greater Cairo area (16 square miles = approximately 40 km 2)
Address correspondence to Ashraf F. Youssef, M.D., Ph.D., 632 Harrison Street, Oak Park, IL 60304. Received 26 April 1995; Revision received 9 November 1995; Accepted 13 November 1995. 145
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mg/m 3). However, Amr et al. (11) estimated that mean respirable lead levels reached 37.2 f-Lg/m3 at one Egyptian daily newspaper's printing facilities. It was also found by Abu-Ali (12) in another study involving the printing industry that blood lead levels reached 80 f-Lg/dL with atmospheric levels of 20 to 40 f-Lg/m3. However, in a recent review study (13) in occupationally exposed workers in Germany, it was found that the blood lead level and air lead level were not strongly correlated. An Agency for Toxic Substances and Diseases Registry (ATSDR) report on lead estimated that male reproductive toxicity occurs when the blood lead level reaches 40 to 50 f-Lg/dL (14). In 1992, OSHA suggested that blood lead levels should not exceed 30 f-Lg/dL for workers (males and females) contemplating conception (15). Lead is listed as a direct-acting reproductive toxicant to both developing and mature reproductive systems (16). However, much controversy exists over the effects on target organs, on male endocrine and reproductive function, and on the pathogenic mechanism(s) involved (17). For instance, Coste et al. (18) found no association between reproductive parameters or fertility status and the degree of exposure among battery-factory workers in France. Direct toxic effects of lead on spermatogenesis were demonstrated but without changes in male endocrinologic parameters (8,19-21). On the other hand, McGregor and Mason (22) showed that workmen who were moderately exposed to lead (i.e., mean lead level = 45 f-Lg/dL) showed significant testicular dysfunction, as well as a mild increase in serum FSH and a significant lowering of serum LH. These results were supported by Braunstein et al. (23), who reported decreased libido, decreased levels of plasma testosterone, and a slight decrease in levels of plasma LH after GnRH stimulation in exposed workers, suggesting Leydig cell damage. It has also been suggested that biochemical and chromosomal alterations play important etiologic roles in leadassociated male reproductive dysfunction (24,25). In the current case-eontrol study, the design was based on a strategy to identify an infertile (INF) population that was divided into two subgroups according to the presence of environmental (INF-E) or environmental and occupational exposure (lNF-EO). We matched the infertile group to a fertile group. The fertile (F) population, in whom comparatively minimal elevation in blood lead levels would be expected, represented the normal overexposure of Cairo residents (E) and was used as the control group. The two matching fertile subgroups were F-E and F-EO. In addition, the strategy of the study was to examine the interaction between two main variables: first the degree of exposure [environmental (E) vs. environmental! occupational (EO)] and second, the fertility status [fertile (F) vs. infertile (lNF)]. Blood lead level was used as a
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biologic index for recent exposure, and semen lead level was used as an indicator for the direct exposure of reproductive tissue. Subtle changes in fertility status were evaluated through an assessment of semen parameters and hormones. Thus, chemical biomarkers of exposure (lead in blood and in semen) were compared with endocrinologic/biologic biomarkers (total testosterone, LH, and FSH) and semen parameters (volume, count, motility, and abnormal forms).
MATERIALS AND METHODS Fifty-five Egyptian (Caucasian), urban males, aged 20 to 40 years, from the same socioeconomic background, from greater Cairo, residing in a 16-square-mile area, were the subjects in this study. Thirty infertile (INF) subjects were selected from among the patients at the Kasr El Aini Venereal Diseases Outpatient Clinic, Cairo University, Cairo, Egypt, complaining of infertility, which was defined as the inability of their female sexual partners to achieve pregnancy over the course of at least 2 years in the presence of a continuous sexual relationship and without any diagnosed cause of infertility or ovulation disorder. All subjects were informed of the scope of the study and of the procedures involved, and all freely gave their informed consent to participate in the study. Each subject provided a full medical history including age, residence, special habits (i.e., smoking cigarettes and/or hashish, alcohol intake, and drug use), history of surgical procedures particularly in the lower abdomen, and daily method of transportation (e.g., motor bike vs. car, bike, or public transportation). Occupational history included nature of work, daily duration of work (including any additional part-time activity). Sexual history include duration of marriage, frequency of intercourse, and number of children. Each subject underwent general and local clinical examinations, and gave blood samples (5 to 10 mL) by venipuncture and a semen sample by masturbation after a period of abstinence from sexual activity for at least 3 d into acid-washed glass tubes and vials, respectively. Patients with systemic diseases, such as diabetes mellitus, and bilharzia (i.e., endemic shistosomiasis), or with chronic local conditions, such as a mass in the vas deferens, bilateral varicocele grade I, or unilateral grade II, or with a history of using drugs known to be toxic to the reproductive system or of marijuana use were excluded from the study. No subject with a history of alcohol use was included in the study. These INF subjects (n = 30) were assigned to one of two groups according to their occupation: group INF-EO (n = 15) were occupationally exposed [drivers (n = 2), painters (n = 5), storage battery workers (n = 2), printers (n = 2), mechanics (n = 2), a cement worker (n = 1), and a smelter (n = 1)]; group INF-E (n = 15) were
Lead and male infertility. E. A. EL-ZOHAIRY
nonoccupationally exposed [government employees (n = 6), clerks (n = 5), an electrician (n = 1), shopkeepers (n = 2), and a security guard (n = 1)]. The rest of the subjects were fertile (F) (n = 25). These subjects were chosen from among those who had children, with the variability in the length of marriage taken into consideration. Fertile (F) subjects were assigned to two groups according to their occupational exposure. Fertile subjects were matched with infertile subjects according to occupation and socioeconomic level, and based on the previously mentioned criteria. For example, fertile printers or storage battery workers from a particular print shop or storage battery factory were paired with their infertile workmates from the same setting with the same work assignments. Subjects from other occupations were volunteers identified by advertisment in the outpatient clinic or by personal contact and were selected to match similar infertile subjects. Group F-EO (n = 15) were occupationally exposed drivers (n = 2), painters (n = 3), storage battery workers (n = 2), printers (n = 5), and mechanics (n = 3). Group F-E (n = 10) were nonoccupationally exposed clerks (n = 4), shopkeepers (n = 2), janitors (n = 3), and an electrician (n = 1), and were considered the control group. Subjects' serum, after performing CBC, was frozen at -20°e. It was tested for renal function (urea and creatinine), liver function [SGOT(AST), SGPT(ALT)], and for LH, FSH, and testosterone. Estimations of LH, FSH, and total testosterone were made using the double antibody test (Diagnostic Products Corporation, Los Angeles, CA) according to instructions on the package insert
ET
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of the kit, and using a gamma counter (EMI Nuclear, Enterprise, NE) (26). Semen analysis (volume, count, percent motility, and percent abnormal forms) was performed according to WHO guidelines (27). Blood and semen samples were centrifuged and diluted with deionized water (l: 1 concentration). Lead levels in both semen and blood were measured using an atomic absorption spectrophotometer [Perkin-Elmer, Model 2380, with a heated graphite furnace (HGF) (28)]. Statistical analyses Nonpaired Student's t-test was used for comparing the means of all variances among the four groups. Data from different groups were pooled when deemed necessary for purposes of comparison and inference of plausible biologic relationships. For example, the pooled fertile groups (F-E and F-EO) were compared with the pooled infertile groups (INF-E and INF-EO). Regression analysis and percentage correlation of the variances were also determined using the Statview™ program (Brainpower, Inc., CA), an Apple Macintosh computer program. Fisher exact test was used to compare the population percentages. Statistical significance was considered at P < 0.01. RESULTS
Table 1 shows the step-wise increase in blood lead levels in all groups. Regression analysis revealed no correlation between blood and semen lead levels in either of the two groups (,.2 = 0.30 and ,.2 = 0.30 in the fertile
Table 1. Blood and semen lead (Pb) levels, hormones (LH, FSH, and T), and semen parameters in four groups of urban Egyptian men F-E (n = 10) Blood Pb (fLg/dL) range Semen Pb (fLg/dL) range LH (mID/mL) range FSH (mID/mL) range Testosterone (ng/mL) range Sperm count (miliion/mL) range Motility (%) range Abnormal forms (%) range Semen volume (mL) range
16.90± 2.55 (6-29) 11.10 ± 1.92 (3.5-20) 6.90 ± 0.55 (3.6-10) 5.48 ± 0.58 (3.5-8.0) 6.66 ± 0.46 (4.7-9) 70.60 ± 0.31 (50-92) 65.00± 5.5 (60-80) 19.60±2.5 (10-30) 4.85 ± 0.73 (2-9)
F-EO (n = 15) a
26.73 ± 2.05 (15-39) 21.93 ± 1.98 a (5-36) 4.70 ± 0.56 a (3-6.6) 6.04 ± 0.68 (4-9)
6.81 ± 0.87 (4.5-9.5) 48.60 ± 0.98 a (25-65) 42.00 ± 7.59 (15-60) 25.47 ± 1.33 (20-35) 4.33 ± 0.43 (2-7)
INF-E (n = 15)
INF-EO (n = 15)
29.30 ± 3.75" (6-46) 19.60 ± 2.40a (7-38) 13.7 ± 3.5 b (3-60) 20.78 ± 4.1 ab (3.6-58) 4.85 ± 0.45 (1.9-8) 17.14 ± 4.74ac (0-53) 33.00 ± 5.82 c (1-70) 42.14 ± 4.21 ac
37.02±4.91 a (15-70) 25.80 ± 3.15" (8-53) 9.86 ± 0.43 b (4.2-20) 13.15 ± 2.02 ab (5.2-33) 4.89 ± 1.21 (1.6-8) 7.20 ± 2.09 ad (0-22) 36.07 ± 3.86 d (15-60) 43.85 ± 1.8ad (30-50) 3.07 ± 0.34 (1-6)
(20-80) 3.40 ± 0.37 (1-6)
aSignificantly different from F-E; bSignificantly different from F-EO (P < 0.01); cbased on n = 10; dbased on n = 13. F-E = Fertile environmentally exposed; F-EO = Fertile environmentally and occupationally exposed; INF-E = Infertile environmentally exposed; INF-EO = Infertile environmentally and occupationally exposed. Values are mean ± SEM.
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and infertile groups, respectively). All levels were significantly different from those of group F-E (considered the control). Semen lead levels recorded in all subjects were always less than blood lead levels. In the F-E group, no correlation was found between blood lead levels and semen lead levels or between blood lead levels and FSH, LH, and testosterone levels. However, a positive correlation (? = 0.70, P < 0.01) was found in the INF-EO group between blood lead and semen lead levels, which was not observed in any other group. Comparing pooled fertile cases with pooled infertile cases showed levels of lead that were 22.82 ± 9.16 vs. 33.18 ± 15.76 fLg/dL in blood and 17.67 ± 8.78 vs. 22.56 ± 11.03 fLg/dL in semen. Statistically significant differences were found between the two groups in blood (P < 0.005) but not in semen. In F-EO, with blood lead levels significantly higher than in F-E (P < 0.005), an inverse correlation was found between blood lead levels and LH levels (r2 = 0.96), and a positive correlation was found with FSH levels (? = 0.58) in serum. In INF-EO, on the other hand, no correlation was found between lead levels in blood and testosterone (r2 = 0.046), FSH (? = 0.062), and LH levels (? = 0.15) in serum. Serum LH level (Table I) in F-E was significantly higher than in F-EO (P < 0.0048), while significantly lower than in INF-EO (P < 0.01) and in INF-E (P < 0.0003). However, INF-E showed the highest increase in LH levels, while there was a lack of significance between
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INF-E and INF-EO. At the same time, there was no significant difference in total serum testosterone levels among the four groups. Serum FSH levels (Table I) were not significantly different between F-E and F-EO, and were significantly higher in INF-E than in F-E (P < 0.0036), while in INF-EO, they were higher than in F-EO (P < 0.0014), indicating that changes in FSH levels are not due to lead exposure. Table 2 shows that the proportion of men with oligospermia « 20 X 106/rnL) was similar in the infertile population INF-E (73.3%), compared to the population with occupational lead exposure (66.7%, P NS). However, Table 1 shows a significant decrease in sperm count among the INF-EO (i.e., compared with INF-E; 7.20 vs. 17.14 x 106 , respectively). Table 2 shows an ascending increase in the percentage of abnormal forms (> 35%), which was 70% in INF-E (n = 10) vs. 92.3% in INF-EO (n = 13) and was statistically significant. Statistically significant differences were observed for the sperm count and motility based on fertility status. Semen volume was> 1.5 mL in all four groups, which was acceptable according to WHO guidelines (27). Regression analysis between age and semen parameters showed no correlation with semen volume, motility, and abnormal forms (? = 0.1, 0.004, and 0.004, respectively). No clinical toxic manifestations (e.g., neurologic manifestations, gastrointestinal ailments), CBC, or liver and renal function abnormalities were observed among the four groups.
Table 2. Population frequency, percent of subjects with changes in semen parameters and mean values of blood and semen lead levels in four groups of adult Egyptian males F-EO (n = 15)
F-E (n = 10) Sperm count million/mL (accepted = 20/mL)
Motility (%) (accepted> 50)
Abnormal forms (%) (accepted < 40)
Volume (accepted> 1.5 mL)
INF-E (n
=
15)
abd
%
9 2 3 I
60 13.3 20 6.7
%
n
%
n
%
<10 <20 <40 >40
0 0 0 10
0 0 0 100
0 0 4 II
0 0 26.7 73.3
n
%
nab
0 33.3 26.7 40
I 3 2 4
10 30 20 40 %
n
%
n
%
<20 <35 ",,50 >50
0 0 0 10
0 0 0 100
%
n
%
n
%
nab
4 6 0 0
40 60 0 0
4 11 0 0
26.6 73.3 0 0
0 3 4 3
%
n
20 20 60
2 4 9
<20 <35 ",,50 >50 mL <2 <5 >5
n
2 2 6
0 5 4 6
%
13 27 60
%
0 30 40 30
INF-EO (n n
=
15)
abe
%
6 4 3 2
40 26.7 20 13.3
nab
I 5 2 5 n abc
0 I 7 5
%
7.7 38.5 15.4 38.5 %
0 7.7 53.8 38.5
n
%
n
%
4 10 I
26.7 66.7 6.7
4 11 0
26.7 73.3 0
aSignificantly different from F-E; bSignificantly different from F-EO; CSignificantly different from INF-E using Fisher exact test. dFive patients were azoospermic; e two patients were azoospermic. F-E = Fertile environmentally exposed; F-EO = Fertile environmentally and occupationally exposed; INF-E = Infertile environmentally exposed; INF-EO = Infertile environmentally and occupationally exposed.
Lead and male infertility.
DISCUSSION
Allowable safe concentrations of toxic pollutants are still a debatable issue in developing countries where there is wide variation in human susceptibility and tolerance, which depend on general human health and other environmental factors (29). Burimovitz and his colleagues (30) suggested that lead in semen is a more accurate index of the degree of reproductive exposure than lead in blood. In the current study, blood lead levels were always higher than related semen lead levels, but a correlation existed only in INF-EO (i.e., the highest levels) between the two parameters (,-2 = 0.7, P < 0.01). In addition, the lack of significance in semen lead levels between INF-E and INF-EO vs. levels in blood, which showed high significance, invalidates the use of lead level in semen as a biologic indicator for lead exposure. This postulation also matches the experimental results of Platchy et al. (31). Pooling of fertile Egyptian men (F-E and F-EO) as a group (F) produced a mean semen lead level of 17.67 J..lg/dL , which was about threefold higher than the values recorded in Europe [i.e., 5.2 J..lg/dL (8)]. However, we recommend the subclassification provided in our study as a more precise comparison methodology. Lead levels, both in semen and in blood, were always higher in the infertile population than in the fertile. Comparing pooled fertile cases with pooled infertile cases showed significant differences in lead levels between the two groups in blood (P < 0.005), but was only significant at P < 0.05 in semen. Regression analysis showed no correlation between blood and semen lead levels in either of the two groups (,-2 = 0.30 in the fertile and infertile groups). These results showed an absence of any correlation between blood and semen lead levels, which was also observed by Xu et al. (32). A positive relationship was demonstrated between low-dose environmental exposure to lead and unexplained poor sperm parameters in males and/or a decrease in male fertility (33,34). Wyrobek et al. (35), suggested that the sperm morphology is the most sensitive parameter to use in studying changes in spermatogenesis caused by toxic substances. Morphology is also a good indicator for the state of the testicular germinal epithelium (36). Comparison of sperm count, percent abnormal forms, and percent motility demonstrated statistically significant differences based on fertility status. However, the only statistically significant difference between the two infertile groups (i.e., INF-E and INF-EO) was in the percent abnormal forms. These changes suggested morphology as a more sensitive index to the effect of lead than other semen parameters. This observation may be
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the result of an added effect of lead to infertility on sperm, particularly at high levels. In the current study, several changes that were observed are summarized in Tables 1 and 2. First, there was a significant decrease in sperm count among all groups compared with F-E. Second, there was a significant decrease in F-EO compared with F-E (P < 0.001) but the maximum decrease was in INF-EO (P < 0.001), which was also significantly different from INF-E. However, there was no effect on volume, which may be due to the absence of any effect on the vas or the prostate; this finding is contradictory to results demonstrated in experimental animals (37). Decreased sperm chromatin stability in men chronically exposed to lead has been demonstrated previously (25). Wang et al. (38) explained that moderate occupational exposure, close to the present occupational standards, causes qualitative changes in semen parameters, including direct and indirect effects of lead on accessory genital function and sperm maturation. In a recent study involving rats (39), it was demonstrated that at 30 J..lg/dL lead there is a reduction in the ability of sperm to penetrate or fertilize the egg of nonexposed females in the absence of ultrastructural changes. This concurred with a study in mice by Johansson and Wide (40). In the current study, differences in motility percent did not indicate an overt direct relationship with the level of lead exposure. However, Chia et al. (41) have demonstrated that high blood lead levels in infertile men are associated with asthenspermia (P < 0.003). Total testosterone levels in serum were not significantly affected by exposure to lead among the four groups. This could be explained by the lack of sensitivity of total testosterone to hormonal changes. Free serum testosterone (2 to 3% of total testosterone) is a more sensitive index, but was not measured in the current study. Furthermore, the relevance of the intratesticular testosterone to spermatogenesis as opposed to serum testosterone was also emphasized by Jaffe and Yen (42). This hypothesis was supported by Bellinger (16), who hypothesized that lead is not directly toxic to testicular tissue, but rather affects the androgenic control of the testis, impairing spermatogenesis and intratesticular testosterone levels. Changes in FSH levels did not correlate with blood lead levels. This may be explained by the biologic variability and the higher FSH levels that occur in INF-E, and may show that there is no direct effect of lead on FSH levels. On the other hand, individual variability and the bioavailability of lead in cases of infertility may explain the variability in LH levels when there are high blood lead levels in INF-EO vs. low blood lead levels in F-EO.
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The current study involved subjects with the highest concentration of lead in blood, which were similar to the recorded values in other studies in Egypt [e.g., (9,11)]. Furthermore, this study, to our knowladge, is the first to measure the lead levels in semen of an Egyptian population and yet recorded the highest lead level in semen in a human population. We excluded sample contamination as a possible cause of the recorded high values for lead in semen since identical precautions were taken for both blood and semen in sample preparations, in addition to the observed consistency in the standard error of the mean across samples. Similar studies have been conducted in developed (European or Asian) countries [e.g., (18,21,31,32,39,40)]. However, the difference in the levels of environmental and occupational exposure and the much more stringent standards and environmental regulations may explain, at least in part, the observed differences in levels from the current study. Semen lead levels in F-E Egyptian males (i.e., 11.1 ± 0.92 fLg/dL) matched those of European INF-E in the JockenhOvel et al. (8) study (i.e., 11.18 ± 0.62 fLg/dL). This comparison reflects the difference in levels of exposure, and may imply that there is no strong correlation between infertility and the level of lead in semen and/or there is a confounding effect due to other factors. Further research is needed to address this possibility. We agree with JockenhOvel et al. (8) that the presence of high lead levels in blood and semen in an infertile vs. fertile population cannot explain the infertility observed. However, if lead has no effect on fertility, then lead levels in F-E should not be different from those in INF-E, which is obviously not the case. Future studies are needed that apply the current subclassification to larger-sized random populations and that include baseline measurements of blood and semen lead levels taken prior to subjects' employment in various industries, with periodic followup throughout the period of employment. Acknowledgments - We greatly acknowledge the editorial review of the manuscript by Ms. Debra A. DePalma. This work is based in part on a Ph.D. dissertation at Cairo University, Cairo, Egypt, 1994, and was presented, in part, at the Society of Toxicology meeting, New Orleans, 1993.
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5.
6. 7.
8.
9. 10.
II.
12. 13.
14. 15.
16. 17.
18.
19.
20.
21.
22.
REFERENCES 23. I. UNEP and WHO. Urban air pollution 1973-1980. Global environmental monitoring systems (GEMS). New York: United Nations Environmental Program (UNEP) and Geneva: World Health Organization; 1984. 2. UNEP and WHO. Assessment of urban air quality. Report on the results of the WHOIUNEP program on health-related environmental monitoring. New York: United Nations Environnmental Program (UNEP) and Geneva: World Health Organization; 1988. 3. Nasralla M. Lead in urban environment. Egypt J Occup Med. 1984;8:53-63. 4. Environmental Protection Agency. Quarterly summary of lead, Phasedown Reproduction Data. Washington, DC: Office of Mobile
24. 25.
26. 27.
Sources, Office of Air and Radiation, US Environmental Protection Agency; 1991. Amr MM, Zagloul MA, Youssef MZ. Biological monitoring of lead in Egypt. Doctoral Thesis, Department of Occupational Medicine, Cairo University, Cairo, Egypt; 1991. Nasralla M. Spatial and seasonal variation in lead in Cairo atmosphere. Environmental pollution (series B). 1986;11 :205. Environmental Protection Agency. Air quality criteria for lead. Washington, DC: U.S. Environmental Protection Agency, Office of Research and Development; 1977. Jockenhovel F, Bals-Pratsch M, Bertram HP, Nieschlag E. Seminal lead and copper in fertile and infertile men. Andrologia. 1990; 22:503-11. Emara AM, Zagloul AM, Zayet HH. Auto-exhaust pollution in Cairo. Egypt J Occup Med. 1990;14:1-12. EI Samra G, Emara H, EI Sobky M. The extent of environmental pollution from motor vehicles and its biological indices, Doctoral Thesis, Department of Occupational Medicine, Cairo University; 1992. Amr MM, El Batanoni M, Osman A, Allam M, EI-Dessouky M. Neuropsychiatric and hematological manifestations in workers exposed to lead. Egypt J Occup Med. 1986;10:211-9. Abu Ali. Correlation between environmental and biological levels of lead in printing industry. Egypt J Occp Med. 1986;10:183-93. Kentner M, Fischer T. Lead exposure in starter battery production: investigation of the correlation between air lead and blood lead levels. Int Arch Occup Environ Health. 1994;66:223-8. ATSDRffP-88/l7 and EPA toxicological profile for lead (June 1990). Syracuse Research Corp. OSHA. Assessing the relationship between environmental lead concentrations and adult blood lead levels. Cambridge, MA, 02138 11/3-18; 1992. Bellinger D. Teratogen update: lead. Teratology. 1994;50:367-73. Mann T, Lutwak-Mann C. Male reproductive function and semen. Themes and trends in physiology, biochemistry, and investigative andrology. New York: Springer Verlag; 1986. Coste J, Mandereau L, Pessione F. Lead exposed workmen and fertility: a cohort study on 354 subjects. Eur J Epidemiol. 1991;7: 154-8. Molinini R, Asseanto G, Galiardi T, Paci, C. Lead effects on male fertility in battery workers. Communication to the International Occupational J Health Congress, Cairo; 1981. Lancranjan I, Popescu HI, Favanescu G, Klepsch I, Serbanescu M. Reproductive ability of workmen occupationally exposed to lead. Arch Environ Health. 1975;30:396-401. Assennato G, Paci C, Baser M, Molinini R, Candela RG, Altamura BM, Giorgino R. Sperm count suppression without endocrine dysfunction in lead exposed men. Arch Environ Health. 1987;42: 124-7. McGregor AJ, Mason MJ. Chronic occupational lead exposure and testicular endocrine function. Hum Exp Toxicol. 1990;9:371-6. Braunstein GD, Dahlgren J, Loriaux DL. Hypogonadism in chronically lead-poisoned men. Infertility. 1978;1:33-51. Thomas JA, Borgan WC III. Some actions of lead on the sperm and on the male reproductive system. Am J Ind Med. 1983;4: 127-34. Wildet K, Ellison R, Berlin M. Effects of occupational exposure to lead on sperm and semen. In Clarkson TW, Nordberg GF, Sager PR, eds. Reproductive and developmental toxicity of metals. New York: Plenum; 1983:279-300. Jaffe BM, Buhrman MR. Methods of hormone radioimmunoassay. New York: Academic Press; 1974. World Health Organization (WHO). Laboratory manual for examination of human semen and semen-cervical mucus interaction. 2nd
Lead and male infertility. E. A. EL-ZOHAIRY ed. Cambridge: Cambridge University; 1987:6-7. 28. Brown AA, Halls DJ, Taylor A. Atomic spectrophotometry update-clinical and biological materials; Food and beverages. J Anal Atomic Spec!. 1988;3:45. 29. World Health Organization (WHO). Lead. Environmental Health Criteria on Inorganic Lead. No.3; 1977. 30. Butrimvitz GP, Sharlip I, Lo R. Extremely low seminal lead concentrations and male fertility. Clin Chem Acta. 1983;135:229-31. 31. Plechaty MM, Noll B, Sunderman FW Jr. Lead concentrations in semen of healthy men without occupational exposure to lead. Ann Clin Lab Sci. 1977;7:515-8. 32. Xu B, Chia E, Ong CN. Concentration of cadmium, lead, selenium and zinc in human blood and seminal plasma. Bioi Trace Elem Res. 1994;40:49-57. 33. Saaranen M, Kantola S, Vanha T. Lead, magnesium, selenium and zinc in human seminal fluid: comparison with semen parameters and fertility. Hum Reprod. 1989;2:475-9. 34. Lee S, Chia E, Ong C. Blood concentration of lead, cadmium, mercury, zinc and copper and human semen parameters. Arch Androl. 1992;29:177-83. 35. Wyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kepp RW Jr, Letz G, Mailing HV, Tophan JC, Whorton MD. An evaluation of
36.
37. 38.
39.
40. 41.
42.
ET
AL.
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human sperm as indicators of chemically-induced alterations of spermatogenic function. Mutat Res. 1983;115:73-148. Jeyendran RS, Van der Yen HH, Parez Prez M, Crabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 1984;70:219-27. Chowdbury AR, Dewan A, Gandhi DN. Toxic effects of lead on the testes of rats. Biomed Biochem Acta. 1984; I :95-100. Wang L, Ong C, Foo S. LDH-X in human seminal plasma as an indicator of the functional integrity of spermatozoa. J Am Coli Health. 1992;41:115-9. Sokol R, Okuda H, Nagler H, Berman N. Lead exposure in vivo alters the fertility potential of sperm in vitro. Toxicol Appl Pharmacol. 1994;124:310-6. Johansson L, Wide M. Long-term exposure of the male mouse to lead: effects on fertility. Environ Res. 1986;41:481-7. Chia SE, Ong C, Lee ST, Tsakak FH. Blood concentrations of lead, cadmium, mercury, zinc and copper and human semen parameters. Arch Androl. 1992;29: 177-83. Jaffe R, Yen S. The hypothalamic-pituitary-testicular axis. In Robert B, Samuel C, eds. Reproductive endocrinology. London: WB Saunders Company; 1991.
0890-6238(95)02057-8
REPRODUCTIVE HAZARDS OF LEAD EXPOSURE AMONG URBAN EGYPTIAN MEN EMAN
A. EL-ZOHAIRY,* ASHRAF F. YOUSSEF,* SAID M. ABUL-NASR,*
IBRAHIM M. FAHMyt DAWLAT SALEM,:j: AZIZA K. KAHIL,* and MAHMOUD K. MADKOUR§ Departments of *Forensic Medicine and Toxicology, :j:Biochemistry, §Pharmacology, and tVenereal Diseases, Cairo University, Cairo, Egypt Abstract - Fifty-five urban Egyptian males, aged 20-40, were assigned to two main groups to study the effects of their exposure to lead (Pb). Group I, infertile men (INF, n = 30), was divided into environmentally exposed (INF-E, n 15) and environmentally and occupationally exposed (INF-EO, n 15). A matching group (II) of fertile men (F, n = 25) was divided into fertile, environmentally exposed (F-E, n = 10), which was the control group, and fertile, environmentally and occupationally exposed (F-EO, n = 15). Semen parameters (i.e., count, morphology, motility, and volume), blood and semen Pb levels, and reproductive hormonal indices (i.e., serum testosterone, FSH, and LH) were measured in all subjects. Lead levels were always higher in blood than semen. Semen lead levels were significantly higher in all groups vs. the control (F-E) group. While no changes were observed in testosterone levels across groups, variable effects on LH and FSH levels were observed. Infertile-EO subjects showed a definite pattern of impaired semen parameters in comparison with infertile-E. No abnormalities were detected in hematologic, hepatic or renal function.
=
=
Key Words: lead; infertility; males; semen parameters; gonadotropins; occupational; environmental.
INTRODUCTION
include cement, chemicals, fertilizers, iron, steel, nonferrous metallurgy (lead and zinc), asbestos, and electric power, in addition to other minor sources of air pollution (e.g., printing and smelting). Furthermore, the lead constituent of dustfall over southern Cairo has reached 1,652 f.Lg/g of dust (40 to 1652 range) and its solubility has been estimated to be at least 23 to 53%, which increases its bioavailability in comparison with the lead in other countries (e.g., 10 to 20% in the UK) due to differences in atmospheric constituents and conditions (6). A significant correlation between a decrease in lead levels in gasoline and a decrease in blood lead levels has been shown (7). In East Germany, a highly significant (P < 0.0002) decrease in lead levels in blood and semen over 2 years was associated with a decrease in the lead level in gasoline (8). In Cairo, the blood lead level among traffic policemen has been estimated at 39.16 ± 2.12, the level for drivers at 34.61 ± 2.82, and the level for gas station mechanics at 36.6 ± 3.28 f.Lg/dL (9). El Samra and colleagues (10) measured mean blood lead levels among housewives, female rural employees, male rural employees, drivers, and policemen to be 11.2, 16.9, 17.5, 28.2, and 27.3 f.Lg/dL, respectively. Nasralla (3) estimated blood lead levels in traffic policemen to be as high as 39 f.Lg/dL and 62.7 f.Lg/dL in moderately and heavily exposed groups, respectively. It is worth mentioning that the threshold limit value (TLV) in Egypt is 0.20 mg/m 3 , which is close to that of the U.S. (0.15
Cairo, Egypt is considered one of the most polluted cities in the world (1,2). The lead concentration in the atmosphere of Cairo was measured at 10 f.Lg/m 3 in 1980 and 4.5 f.Lg/m 3 in 1984 (3), both of which exceed World Health Organization (WHO) guidelines of 0.5 to 1 f.Lg/m 3 (1,2). The U.S. Environmental Protection Agency (EPA) (4) has estimated that globally approximately 90% of the lead entering the atmosphere comes from the combustion of leaded gasoline. In Cairo, leaded gasoline has an average lead of 0.2 to 0.4 gIL added as tetra-ethyl lead, and is used without proper controls on car exhaust quality. Like leaded gasoline, leaded paints, which have inorganic lead compounds of more than 40% by weight, are also still in common use. Furthermore, there is an outdated network of lead pipes in use for the water supply, and the lead level officially allowed in Egyptian water is 0.1 mglL, which is double the level recommended by the WHO (5). Other topographic and environmental conditions contribute to high lead levels in the Cairo environment. For instance, industry activities in the greater Cairo area (16 square miles = approximately 40 km 2)
Address correspondence to Ashraf F. Youssef, M.D., Ph.D., 632 Harrison Street, Oak Park, IL 60304. Received 26 April 1995; Revision received 9 November 1995; Accepted 13 November 1995. 145
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mg/m 3). However, Amr et al. (11) estimated that mean respirable lead levels reached 37.2 f-Lg/m3 at one Egyptian daily newspaper's printing facilities. It was also found by Abu-Ali (12) in another study involving the printing industry that blood lead levels reached 80 f-Lg/dL with atmospheric levels of 20 to 40 f-Lg/m3. However, in a recent review study (13) in occupationally exposed workers in Germany, it was found that the blood lead level and air lead level were not strongly correlated. An Agency for Toxic Substances and Diseases Registry (ATSDR) report on lead estimated that male reproductive toxicity occurs when the blood lead level reaches 40 to 50 f-Lg/dL (14). In 1992, OSHA suggested that blood lead levels should not exceed 30 f-Lg/dL for workers (males and females) contemplating conception (15). Lead is listed as a direct-acting reproductive toxicant to both developing and mature reproductive systems (16). However, much controversy exists over the effects on target organs, on male endocrine and reproductive function, and on the pathogenic mechanism(s) involved (17). For instance, Coste et al. (18) found no association between reproductive parameters or fertility status and the degree of exposure among battery-factory workers in France. Direct toxic effects of lead on spermatogenesis were demonstrated but without changes in male endocrinologic parameters (8,19-21). On the other hand, McGregor and Mason (22) showed that workmen who were moderately exposed to lead (i.e., mean lead level = 45 f-Lg/dL) showed significant testicular dysfunction, as well as a mild increase in serum FSH and a significant lowering of serum LH. These results were supported by Braunstein et al. (23), who reported decreased libido, decreased levels of plasma testosterone, and a slight decrease in levels of plasma LH after GnRH stimulation in exposed workers, suggesting Leydig cell damage. It has also been suggested that biochemical and chromosomal alterations play important etiologic roles in leadassociated male reproductive dysfunction (24,25). In the current case-eontrol study, the design was based on a strategy to identify an infertile (INF) population that was divided into two subgroups according to the presence of environmental (INF-E) or environmental and occupational exposure (lNF-EO). We matched the infertile group to a fertile group. The fertile (F) population, in whom comparatively minimal elevation in blood lead levels would be expected, represented the normal overexposure of Cairo residents (E) and was used as the control group. The two matching fertile subgroups were F-E and F-EO. In addition, the strategy of the study was to examine the interaction between two main variables: first the degree of exposure [environmental (E) vs. environmental! occupational (EO)] and second, the fertility status [fertile (F) vs. infertile (lNF)]. Blood lead level was used as a
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biologic index for recent exposure, and semen lead level was used as an indicator for the direct exposure of reproductive tissue. Subtle changes in fertility status were evaluated through an assessment of semen parameters and hormones. Thus, chemical biomarkers of exposure (lead in blood and in semen) were compared with endocrinologic/biologic biomarkers (total testosterone, LH, and FSH) and semen parameters (volume, count, motility, and abnormal forms).
MATERIALS AND METHODS Fifty-five Egyptian (Caucasian), urban males, aged 20 to 40 years, from the same socioeconomic background, from greater Cairo, residing in a 16-square-mile area, were the subjects in this study. Thirty infertile (INF) subjects were selected from among the patients at the Kasr El Aini Venereal Diseases Outpatient Clinic, Cairo University, Cairo, Egypt, complaining of infertility, which was defined as the inability of their female sexual partners to achieve pregnancy over the course of at least 2 years in the presence of a continuous sexual relationship and without any diagnosed cause of infertility or ovulation disorder. All subjects were informed of the scope of the study and of the procedures involved, and all freely gave their informed consent to participate in the study. Each subject provided a full medical history including age, residence, special habits (i.e., smoking cigarettes and/or hashish, alcohol intake, and drug use), history of surgical procedures particularly in the lower abdomen, and daily method of transportation (e.g., motor bike vs. car, bike, or public transportation). Occupational history included nature of work, daily duration of work (including any additional part-time activity). Sexual history include duration of marriage, frequency of intercourse, and number of children. Each subject underwent general and local clinical examinations, and gave blood samples (5 to 10 mL) by venipuncture and a semen sample by masturbation after a period of abstinence from sexual activity for at least 3 d into acid-washed glass tubes and vials, respectively. Patients with systemic diseases, such as diabetes mellitus, and bilharzia (i.e., endemic shistosomiasis), or with chronic local conditions, such as a mass in the vas deferens, bilateral varicocele grade I, or unilateral grade II, or with a history of using drugs known to be toxic to the reproductive system or of marijuana use were excluded from the study. No subject with a history of alcohol use was included in the study. These INF subjects (n = 30) were assigned to one of two groups according to their occupation: group INF-EO (n = 15) were occupationally exposed [drivers (n = 2), painters (n = 5), storage battery workers (n = 2), printers (n = 2), mechanics (n = 2), a cement worker (n = 1), and a smelter (n = 1)]; group INF-E (n = 15) were
Lead and male infertility. E. A. EL-ZOHAIRY
nonoccupationally exposed [government employees (n = 6), clerks (n = 5), an electrician (n = 1), shopkeepers (n = 2), and a security guard (n = 1)]. The rest of the subjects were fertile (F) (n = 25). These subjects were chosen from among those who had children, with the variability in the length of marriage taken into consideration. Fertile (F) subjects were assigned to two groups according to their occupational exposure. Fertile subjects were matched with infertile subjects according to occupation and socioeconomic level, and based on the previously mentioned criteria. For example, fertile printers or storage battery workers from a particular print shop or storage battery factory were paired with their infertile workmates from the same setting with the same work assignments. Subjects from other occupations were volunteers identified by advertisment in the outpatient clinic or by personal contact and were selected to match similar infertile subjects. Group F-EO (n = 15) were occupationally exposed drivers (n = 2), painters (n = 3), storage battery workers (n = 2), printers (n = 5), and mechanics (n = 3). Group F-E (n = 10) were nonoccupationally exposed clerks (n = 4), shopkeepers (n = 2), janitors (n = 3), and an electrician (n = 1), and were considered the control group. Subjects' serum, after performing CBC, was frozen at -20°e. It was tested for renal function (urea and creatinine), liver function [SGOT(AST), SGPT(ALT)], and for LH, FSH, and testosterone. Estimations of LH, FSH, and total testosterone were made using the double antibody test (Diagnostic Products Corporation, Los Angeles, CA) according to instructions on the package insert
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of the kit, and using a gamma counter (EMI Nuclear, Enterprise, NE) (26). Semen analysis (volume, count, percent motility, and percent abnormal forms) was performed according to WHO guidelines (27). Blood and semen samples were centrifuged and diluted with deionized water (l: 1 concentration). Lead levels in both semen and blood were measured using an atomic absorption spectrophotometer [Perkin-Elmer, Model 2380, with a heated graphite furnace (HGF) (28)]. Statistical analyses Nonpaired Student's t-test was used for comparing the means of all variances among the four groups. Data from different groups were pooled when deemed necessary for purposes of comparison and inference of plausible biologic relationships. For example, the pooled fertile groups (F-E and F-EO) were compared with the pooled infertile groups (INF-E and INF-EO). Regression analysis and percentage correlation of the variances were also determined using the Statview™ program (Brainpower, Inc., CA), an Apple Macintosh computer program. Fisher exact test was used to compare the population percentages. Statistical significance was considered at P < 0.01. RESULTS
Table 1 shows the step-wise increase in blood lead levels in all groups. Regression analysis revealed no correlation between blood and semen lead levels in either of the two groups (,.2 = 0.30 and ,.2 = 0.30 in the fertile
Table 1. Blood and semen lead (Pb) levels, hormones (LH, FSH, and T), and semen parameters in four groups of urban Egyptian men F-E (n = 10) Blood Pb (fLg/dL) range Semen Pb (fLg/dL) range LH (mID/mL) range FSH (mID/mL) range Testosterone (ng/mL) range Sperm count (miliion/mL) range Motility (%) range Abnormal forms (%) range Semen volume (mL) range
16.90± 2.55 (6-29) 11.10 ± 1.92 (3.5-20) 6.90 ± 0.55 (3.6-10) 5.48 ± 0.58 (3.5-8.0) 6.66 ± 0.46 (4.7-9) 70.60 ± 0.31 (50-92) 65.00± 5.5 (60-80) 19.60±2.5 (10-30) 4.85 ± 0.73 (2-9)
F-EO (n = 15) a
26.73 ± 2.05 (15-39) 21.93 ± 1.98 a (5-36) 4.70 ± 0.56 a (3-6.6) 6.04 ± 0.68 (4-9)
6.81 ± 0.87 (4.5-9.5) 48.60 ± 0.98 a (25-65) 42.00 ± 7.59 (15-60) 25.47 ± 1.33 (20-35) 4.33 ± 0.43 (2-7)
INF-E (n = 15)
INF-EO (n = 15)
29.30 ± 3.75" (6-46) 19.60 ± 2.40a (7-38) 13.7 ± 3.5 b (3-60) 20.78 ± 4.1 ab (3.6-58) 4.85 ± 0.45 (1.9-8) 17.14 ± 4.74ac (0-53) 33.00 ± 5.82 c (1-70) 42.14 ± 4.21 ac
37.02±4.91 a (15-70) 25.80 ± 3.15" (8-53) 9.86 ± 0.43 b (4.2-20) 13.15 ± 2.02 ab (5.2-33) 4.89 ± 1.21 (1.6-8) 7.20 ± 2.09 ad (0-22) 36.07 ± 3.86 d (15-60) 43.85 ± 1.8ad (30-50) 3.07 ± 0.34 (1-6)
(20-80) 3.40 ± 0.37 (1-6)
aSignificantly different from F-E; bSignificantly different from F-EO (P < 0.01); cbased on n = 10; dbased on n = 13. F-E = Fertile environmentally exposed; F-EO = Fertile environmentally and occupationally exposed; INF-E = Infertile environmentally exposed; INF-EO = Infertile environmentally and occupationally exposed. Values are mean ± SEM.
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and infertile groups, respectively). All levels were significantly different from those of group F-E (considered the control). Semen lead levels recorded in all subjects were always less than blood lead levels. In the F-E group, no correlation was found between blood lead levels and semen lead levels or between blood lead levels and FSH, LH, and testosterone levels. However, a positive correlation (? = 0.70, P < 0.01) was found in the INF-EO group between blood lead and semen lead levels, which was not observed in any other group. Comparing pooled fertile cases with pooled infertile cases showed levels of lead that were 22.82 ± 9.16 vs. 33.18 ± 15.76 fLg/dL in blood and 17.67 ± 8.78 vs. 22.56 ± 11.03 fLg/dL in semen. Statistically significant differences were found between the two groups in blood (P < 0.005) but not in semen. In F-EO, with blood lead levels significantly higher than in F-E (P < 0.005), an inverse correlation was found between blood lead levels and LH levels (r2 = 0.96), and a positive correlation was found with FSH levels (? = 0.58) in serum. In INF-EO, on the other hand, no correlation was found between lead levels in blood and testosterone (r2 = 0.046), FSH (? = 0.062), and LH levels (? = 0.15) in serum. Serum LH level (Table I) in F-E was significantly higher than in F-EO (P < 0.0048), while significantly lower than in INF-EO (P < 0.01) and in INF-E (P < 0.0003). However, INF-E showed the highest increase in LH levels, while there was a lack of significance between
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INF-E and INF-EO. At the same time, there was no significant difference in total serum testosterone levels among the four groups. Serum FSH levels (Table I) were not significantly different between F-E and F-EO, and were significantly higher in INF-E than in F-E (P < 0.0036), while in INF-EO, they were higher than in F-EO (P < 0.0014), indicating that changes in FSH levels are not due to lead exposure. Table 2 shows that the proportion of men with oligospermia « 20 X 106/rnL) was similar in the infertile population INF-E (73.3%), compared to the population with occupational lead exposure (66.7%, P NS). However, Table 1 shows a significant decrease in sperm count among the INF-EO (i.e., compared with INF-E; 7.20 vs. 17.14 x 106 , respectively). Table 2 shows an ascending increase in the percentage of abnormal forms (> 35%), which was 70% in INF-E (n = 10) vs. 92.3% in INF-EO (n = 13) and was statistically significant. Statistically significant differences were observed for the sperm count and motility based on fertility status. Semen volume was> 1.5 mL in all four groups, which was acceptable according to WHO guidelines (27). Regression analysis between age and semen parameters showed no correlation with semen volume, motility, and abnormal forms (? = 0.1, 0.004, and 0.004, respectively). No clinical toxic manifestations (e.g., neurologic manifestations, gastrointestinal ailments), CBC, or liver and renal function abnormalities were observed among the four groups.
Table 2. Population frequency, percent of subjects with changes in semen parameters and mean values of blood and semen lead levels in four groups of adult Egyptian males F-EO (n = 15)
F-E (n = 10) Sperm count million/mL (accepted = 20/mL)
Motility (%) (accepted> 50)
Abnormal forms (%) (accepted < 40)
Volume (accepted> 1.5 mL)
INF-E (n
=
15)
abd
%
9 2 3 I
60 13.3 20 6.7
%
n
%
n
%
<10 <20 <40 >40
0 0 0 10
0 0 0 100
0 0 4 II
0 0 26.7 73.3
n
%
nab
0 33.3 26.7 40
I 3 2 4
10 30 20 40 %
n
%
n
%
<20 <35 ",,50 >50
0 0 0 10
0 0 0 100
%
n
%
n
%
nab
4 6 0 0
40 60 0 0
4 11 0 0
26.6 73.3 0 0
0 3 4 3
%
n
20 20 60
2 4 9
<20 <35 ",,50 >50 mL <2 <5 >5
n
2 2 6
0 5 4 6
%
13 27 60
%
0 30 40 30
INF-EO (n n
=
15)
abe
%
6 4 3 2
40 26.7 20 13.3
nab
I 5 2 5 n abc
0 I 7 5
%
7.7 38.5 15.4 38.5 %
0 7.7 53.8 38.5
n
%
n
%
4 10 I
26.7 66.7 6.7
4 11 0
26.7 73.3 0
aSignificantly different from F-E; bSignificantly different from F-EO; CSignificantly different from INF-E using Fisher exact test. dFive patients were azoospermic; e two patients were azoospermic. F-E = Fertile environmentally exposed; F-EO = Fertile environmentally and occupationally exposed; INF-E = Infertile environmentally exposed; INF-EO = Infertile environmentally and occupationally exposed.
Lead and male infertility.
DISCUSSION
Allowable safe concentrations of toxic pollutants are still a debatable issue in developing countries where there is wide variation in human susceptibility and tolerance, which depend on general human health and other environmental factors (29). Burimovitz and his colleagues (30) suggested that lead in semen is a more accurate index of the degree of reproductive exposure than lead in blood. In the current study, blood lead levels were always higher than related semen lead levels, but a correlation existed only in INF-EO (i.e., the highest levels) between the two parameters (,-2 = 0.7, P < 0.01). In addition, the lack of significance in semen lead levels between INF-E and INF-EO vs. levels in blood, which showed high significance, invalidates the use of lead level in semen as a biologic indicator for lead exposure. This postulation also matches the experimental results of Platchy et al. (31). Pooling of fertile Egyptian men (F-E and F-EO) as a group (F) produced a mean semen lead level of 17.67 J..lg/dL , which was about threefold higher than the values recorded in Europe [i.e., 5.2 J..lg/dL (8)]. However, we recommend the subclassification provided in our study as a more precise comparison methodology. Lead levels, both in semen and in blood, were always higher in the infertile population than in the fertile. Comparing pooled fertile cases with pooled infertile cases showed significant differences in lead levels between the two groups in blood (P < 0.005), but was only significant at P < 0.05 in semen. Regression analysis showed no correlation between blood and semen lead levels in either of the two groups (,-2 = 0.30 in the fertile and infertile groups). These results showed an absence of any correlation between blood and semen lead levels, which was also observed by Xu et al. (32). A positive relationship was demonstrated between low-dose environmental exposure to lead and unexplained poor sperm parameters in males and/or a decrease in male fertility (33,34). Wyrobek et al. (35), suggested that the sperm morphology is the most sensitive parameter to use in studying changes in spermatogenesis caused by toxic substances. Morphology is also a good indicator for the state of the testicular germinal epithelium (36). Comparison of sperm count, percent abnormal forms, and percent motility demonstrated statistically significant differences based on fertility status. However, the only statistically significant difference between the two infertile groups (i.e., INF-E and INF-EO) was in the percent abnormal forms. These changes suggested morphology as a more sensitive index to the effect of lead than other semen parameters. This observation may be
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the result of an added effect of lead to infertility on sperm, particularly at high levels. In the current study, several changes that were observed are summarized in Tables 1 and 2. First, there was a significant decrease in sperm count among all groups compared with F-E. Second, there was a significant decrease in F-EO compared with F-E (P < 0.001) but the maximum decrease was in INF-EO (P < 0.001), which was also significantly different from INF-E. However, there was no effect on volume, which may be due to the absence of any effect on the vas or the prostate; this finding is contradictory to results demonstrated in experimental animals (37). Decreased sperm chromatin stability in men chronically exposed to lead has been demonstrated previously (25). Wang et al. (38) explained that moderate occupational exposure, close to the present occupational standards, causes qualitative changes in semen parameters, including direct and indirect effects of lead on accessory genital function and sperm maturation. In a recent study involving rats (39), it was demonstrated that at 30 J..lg/dL lead there is a reduction in the ability of sperm to penetrate or fertilize the egg of nonexposed females in the absence of ultrastructural changes. This concurred with a study in mice by Johansson and Wide (40). In the current study, differences in motility percent did not indicate an overt direct relationship with the level of lead exposure. However, Chia et al. (41) have demonstrated that high blood lead levels in infertile men are associated with asthenspermia (P < 0.003). Total testosterone levels in serum were not significantly affected by exposure to lead among the four groups. This could be explained by the lack of sensitivity of total testosterone to hormonal changes. Free serum testosterone (2 to 3% of total testosterone) is a more sensitive index, but was not measured in the current study. Furthermore, the relevance of the intratesticular testosterone to spermatogenesis as opposed to serum testosterone was also emphasized by Jaffe and Yen (42). This hypothesis was supported by Bellinger (16), who hypothesized that lead is not directly toxic to testicular tissue, but rather affects the androgenic control of the testis, impairing spermatogenesis and intratesticular testosterone levels. Changes in FSH levels did not correlate with blood lead levels. This may be explained by the biologic variability and the higher FSH levels that occur in INF-E, and may show that there is no direct effect of lead on FSH levels. On the other hand, individual variability and the bioavailability of lead in cases of infertility may explain the variability in LH levels when there are high blood lead levels in INF-EO vs. low blood lead levels in F-EO.
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The current study involved subjects with the highest concentration of lead in blood, which were similar to the recorded values in other studies in Egypt [e.g., (9,11)]. Furthermore, this study, to our knowladge, is the first to measure the lead levels in semen of an Egyptian population and yet recorded the highest lead level in semen in a human population. We excluded sample contamination as a possible cause of the recorded high values for lead in semen since identical precautions were taken for both blood and semen in sample preparations, in addition to the observed consistency in the standard error of the mean across samples. Similar studies have been conducted in developed (European or Asian) countries [e.g., (18,21,31,32,39,40)]. However, the difference in the levels of environmental and occupational exposure and the much more stringent standards and environmental regulations may explain, at least in part, the observed differences in levels from the current study. Semen lead levels in F-E Egyptian males (i.e., 11.1 ± 0.92 fLg/dL) matched those of European INF-E in the JockenhOvel et al. (8) study (i.e., 11.18 ± 0.62 fLg/dL). This comparison reflects the difference in levels of exposure, and may imply that there is no strong correlation between infertility and the level of lead in semen and/or there is a confounding effect due to other factors. Further research is needed to address this possibility. We agree with JockenhOvel et al. (8) that the presence of high lead levels in blood and semen in an infertile vs. fertile population cannot explain the infertility observed. However, if lead has no effect on fertility, then lead levels in F-E should not be different from those in INF-E, which is obviously not the case. Future studies are needed that apply the current subclassification to larger-sized random populations and that include baseline measurements of blood and semen lead levels taken prior to subjects' employment in various industries, with periodic followup throughout the period of employment. Acknowledgments - We greatly acknowledge the editorial review of the manuscript by Ms. Debra A. DePalma. This work is based in part on a Ph.D. dissertation at Cairo University, Cairo, Egypt, 1994, and was presented, in part, at the Society of Toxicology meeting, New Orleans, 1993.
Volume 10, Number 2, 1996
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14. 15.
16. 17.
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22.
REFERENCES 23. I. UNEP and WHO. Urban air pollution 1973-1980. Global environmental monitoring systems (GEMS). New York: United Nations Environmental Program (UNEP) and Geneva: World Health Organization; 1984. 2. UNEP and WHO. Assessment of urban air quality. Report on the results of the WHOIUNEP program on health-related environmental monitoring. New York: United Nations Environnmental Program (UNEP) and Geneva: World Health Organization; 1988. 3. Nasralla M. Lead in urban environment. Egypt J Occup Med. 1984;8:53-63. 4. Environmental Protection Agency. Quarterly summary of lead, Phasedown Reproduction Data. Washington, DC: Office of Mobile
24. 25.
26. 27.
Sources, Office of Air and Radiation, US Environmental Protection Agency; 1991. Amr MM, Zagloul MA, Youssef MZ. Biological monitoring of lead in Egypt. Doctoral Thesis, Department of Occupational Medicine, Cairo University, Cairo, Egypt; 1991. Nasralla M. Spatial and seasonal variation in lead in Cairo atmosphere. Environmental pollution (series B). 1986;11 :205. Environmental Protection Agency. Air quality criteria for lead. Washington, DC: U.S. Environmental Protection Agency, Office of Research and Development; 1977. Jockenhovel F, Bals-Pratsch M, Bertram HP, Nieschlag E. Seminal lead and copper in fertile and infertile men. Andrologia. 1990; 22:503-11. Emara AM, Zagloul AM, Zayet HH. Auto-exhaust pollution in Cairo. Egypt J Occup Med. 1990;14:1-12. EI Samra G, Emara H, EI Sobky M. The extent of environmental pollution from motor vehicles and its biological indices, Doctoral Thesis, Department of Occupational Medicine, Cairo University; 1992. Amr MM, El Batanoni M, Osman A, Allam M, EI-Dessouky M. Neuropsychiatric and hematological manifestations in workers exposed to lead. Egypt J Occup Med. 1986;10:211-9. Abu Ali. Correlation between environmental and biological levels of lead in printing industry. Egypt J Occp Med. 1986;10:183-93. Kentner M, Fischer T. Lead exposure in starter battery production: investigation of the correlation between air lead and blood lead levels. Int Arch Occup Environ Health. 1994;66:223-8. ATSDRffP-88/l7 and EPA toxicological profile for lead (June 1990). Syracuse Research Corp. OSHA. Assessing the relationship between environmental lead concentrations and adult blood lead levels. Cambridge, MA, 02138 11/3-18; 1992. Bellinger D. Teratogen update: lead. Teratology. 1994;50:367-73. Mann T, Lutwak-Mann C. Male reproductive function and semen. Themes and trends in physiology, biochemistry, and investigative andrology. New York: Springer Verlag; 1986. Coste J, Mandereau L, Pessione F. Lead exposed workmen and fertility: a cohort study on 354 subjects. Eur J Epidemiol. 1991;7: 154-8. Molinini R, Asseanto G, Galiardi T, Paci, C. Lead effects on male fertility in battery workers. Communication to the International Occupational J Health Congress, Cairo; 1981. Lancranjan I, Popescu HI, Favanescu G, Klepsch I, Serbanescu M. Reproductive ability of workmen occupationally exposed to lead. Arch Environ Health. 1975;30:396-401. Assennato G, Paci C, Baser M, Molinini R, Candela RG, Altamura BM, Giorgino R. Sperm count suppression without endocrine dysfunction in lead exposed men. Arch Environ Health. 1987;42: 124-7. McGregor AJ, Mason MJ. Chronic occupational lead exposure and testicular endocrine function. Hum Exp Toxicol. 1990;9:371-6. Braunstein GD, Dahlgren J, Loriaux DL. Hypogonadism in chronically lead-poisoned men. Infertility. 1978;1:33-51. Thomas JA, Borgan WC III. Some actions of lead on the sperm and on the male reproductive system. Am J Ind Med. 1983;4: 127-34. Wildet K, Ellison R, Berlin M. Effects of occupational exposure to lead on sperm and semen. In Clarkson TW, Nordberg GF, Sager PR, eds. Reproductive and developmental toxicity of metals. New York: Plenum; 1983:279-300. Jaffe BM, Buhrman MR. Methods of hormone radioimmunoassay. New York: Academic Press; 1974. World Health Organization (WHO). Laboratory manual for examination of human semen and semen-cervical mucus interaction. 2nd
Lead and male infertility. E. A. EL-ZOHAIRY ed. Cambridge: Cambridge University; 1987:6-7. 28. Brown AA, Halls DJ, Taylor A. Atomic spectrophotometry update-clinical and biological materials; Food and beverages. J Anal Atomic Spec!. 1988;3:45. 29. World Health Organization (WHO). Lead. Environmental Health Criteria on Inorganic Lead. No.3; 1977. 30. Butrimvitz GP, Sharlip I, Lo R. Extremely low seminal lead concentrations and male fertility. Clin Chem Acta. 1983;135:229-31. 31. Plechaty MM, Noll B, Sunderman FW Jr. Lead concentrations in semen of healthy men without occupational exposure to lead. Ann Clin Lab Sci. 1977;7:515-8. 32. Xu B, Chia E, Ong CN. Concentration of cadmium, lead, selenium and zinc in human blood and seminal plasma. Bioi Trace Elem Res. 1994;40:49-57. 33. Saaranen M, Kantola S, Vanha T. Lead, magnesium, selenium and zinc in human seminal fluid: comparison with semen parameters and fertility. Hum Reprod. 1989;2:475-9. 34. Lee S, Chia E, Ong C. Blood concentration of lead, cadmium, mercury, zinc and copper and human semen parameters. Arch Androl. 1992;29:177-83. 35. Wyrobek AJ, Gordon LA, Burkhart JG, Francis MW, Kepp RW Jr, Letz G, Mailing HV, Tophan JC, Whorton MD. An evaluation of
36.
37. 38.
39.
40. 41.
42.
ET
AL.
151
human sperm as indicators of chemically-induced alterations of spermatogenic function. Mutat Res. 1983;115:73-148. Jeyendran RS, Van der Yen HH, Parez Prez M, Crabo BG, Zaneveld LJD. Development of an assay to assess the functional integrity of the human sperm membrane and its relationship to other semen characteristics. J Reprod Fertil. 1984;70:219-27. Chowdbury AR, Dewan A, Gandhi DN. Toxic effects of lead on the testes of rats. Biomed Biochem Acta. 1984; I :95-100. Wang L, Ong C, Foo S. LDH-X in human seminal plasma as an indicator of the functional integrity of spermatozoa. J Am Coli Health. 1992;41:115-9. Sokol R, Okuda H, Nagler H, Berman N. Lead exposure in vivo alters the fertility potential of sperm in vitro. Toxicol Appl Pharmacol. 1994;124:310-6. Johansson L, Wide M. Long-term exposure of the male mouse to lead: effects on fertility. Environ Res. 1986;41:481-7. Chia SE, Ong C, Lee ST, Tsakak FH. Blood concentrations of lead, cadmium, mercury, zinc and copper and human semen parameters. Arch Androl. 1992;29: 177-83. Jaffe R, Yen S. The hypothalamic-pituitary-testicular axis. In Robert B, Samuel C, eds. Reproductive endocrinology. London: WB Saunders Company; 1991.