Clinical Relevance of Vitamin C Among Lead-Exposed Infertile Men

Abstract

Aims and Objectives: A cross-sectional study was designed by targeting 120 male workers occupationally exposed to lead from a battery-manufacturing industry situated at the Patancheru industrial area, Hyderabad, Andhra Pradesh, India, to see the impact of lead on testicular dysfunction with reference to infertility. Further, the study was designed to see the in vivo effect of an antioxidant in the form of vitamin C, prophylactically administered at the dose of 1000 mg/day for five consecutive days in a week for 3 months. Methodology: Blood samples and semen samples were collected from 120 menin the study group exposed to lead, and 120 healthy human subjects, who have no history of exposures to chemicals, were selected as controls for comparison. The mean age of the workers who participated in this study falls in the range of 25-55 years. The semen samples were collected with due consent of the industrial workers to perform the conventional semen analysis and the measure of sperm DNA fragmentation by the comet assay. Results: Industrial workers showed a statistically significant increase in sperm motility (p<0.001), sperm total count (p<0.001), and a statistically significant decrease in abnormal sperm morphology (p<0.001) after vitamin C prophylaxis. The comet assay also showed similar results, where there is a statistically significant decrease in alkaline-labile sites and a statistically significant decrease in the mean tail length of the comet when compared to the control group (p<0.001) after vitamin C prophylaxis. Conclusion: This study leads us to conclude that the lead compound interferes with the testicular function, inducing its activity and also by exerting its effect on sperm DNA, leading to fragmentation. Further, the prophylaxis with antioxidant treatment may offer protection against the reactive oxygen species (ROS)-induced DNA damage, which is a major cause in the etiology of male infertility.

Introduction

Lead is a ubiquitous heavy metal with wide industrial applications, such as battery manufacturing, mining, printing, ceramics, and paints. Lead metal consumption worldwide was estimated to be at 8 million tons, mainly for the production of lead-acid batteries (71%), pigments (12%), rolled extensions (7%), and cable sheathing (3%), by the International Lead and Zinc study group (2007). Both occupational and environmental exposures to lead remain a serious problem in many developing and industrializing countries as well as in some developed countries. The effects of lead are very well known. Exposure to lead and its compounds can damage almost all organs and organ systems, particularly the CNS, kidney, and blood, leading to adverse health implications (Goldstein, 1992; WHO, 1995).

In general, lead exerts its toxic effect by disrupting the balance between the pro-oxidative and antioxidative status of the tissue, leading to oxidative stress. Oxidative stress occurs as a consequence of an imbalance between the production of ROS (reactive oxygen species) and the available antioxidant defense against them (Sikka et al., 1995; Sharma and Agarwal, 1996). The reproductive system, particularly the spermatozoa, is vulnerable to oxidative stress induced by lead, because their plasma membrane and cytoplasm contain large amounts of polyunsaturated fatty acids (Alvarez and Storey, 1995). Earlier studies reveal that lead will affect spermatogenesis, leading to infertility. Blood lead levels (BLLs) of 60 μg/dL may be associated with male infertility (Fisher et al., 1987). Studies in male workers indicate that exposures to lead resulting in BLLs as low as 40 μg/dL may cause a decreased sperm count and abnormal sperm morphology (Lancranjan et al., 1975; Alexander et al., 1996). Several reports indicate that decreased sperm quality and hormonal changes can occur among male workers exposed to lead with BLLs of 30-40 μg/dL (Braunstein et al., 1978; Ng et al., 1991).

There is now a great deal of scientific knowledge about the use of nutritional supplements and possible beneficial effects on both male and female infertility. Many diseases are associated with oxidative stress; this is why the use of antioxidant-rich food or antioxidant food supplements has become immensely popular. These antioxidants include enzymes like superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase; minerals such as selenium, manganese, copper, and zinc; and vitamins such as A, C, and E, beside compounds such as glutathione, uric acid, and flavonoids. These antioxidants protect, prevent, or reduce the extent of oxidative destruction of cellular tissues. Elevated levels of lipid peroxidation products and the simultaneous decline of antioxidant defense mechanisms have been suggested to be harmful through disruption of the membrane lipid and damage of cellular organelles, resulting in oxidative stress (Ifeoma et al., 2009). Diet contains antioxidants in the form of vitamins, of which vitamin C is known to be an effective antioxidant (Agarwal and Prabakaran, 2005) and a chain-breaking compound (Agarwal and Prabakaran, 2005). Many studies conclude that vitamin C enhances sperm quality, by protecting sperm and the DNA from the damage caused by oxidative stress (Dawson et al., 1987; Lutsenko et al., 2002).

Considering lead as an occupational health-hazardous compound and also the protective nature of vitamin C, a modest attempt has been made in this investigation to monitor vitamin C prophylaxis against lead-induced infertility among lead-acid battery-manufacturing industrial workers by adopting a standard conventional semen analysis and the comet assay.

Materials and Methods

Study area

The present study was performed by targeting the lead-acid battery-manufacturing industries situated in the vicinity of industrial development area (IDA), Patancheru, Andhra Pradesh, India.

Study population and design

One hundred twenty industrial workers working in a lead-acid battery-manufacturing industry were targeted for this study. The mean age of industrial workers ranged from 22 to 55, and the mean duration of exposure to lead in the industry was ≥15 years. Similarly, 120 age- and sex-matched unexposed individuals were selected as controls for the study. The industrial workers have been further categorized into three groups based on the nature of the exposure at different working sites in the lead-acid battery-manufacturing units: group I (assembly unit), group II (plate-buffing unit), and group III (dry-charging unit). For vitamin C prophylaxis, only industrial workers were included in this study. Vitamin C prophylaxis has been given under the supervision of a medical practitioner for 3 months [1000 mg/day in two split doses in the tablet form with the brand name Limcee (Glaxo)]. The work has been approved by the Institutional Ethics Committee (University College for Women, Hyderabad, India) and performed as per the ethics norms of biomedical research on humans. The exposed workers selected had a work history of more than 2 years.

Questionnaire

The participants were recruited for reproductive epidemiology by following a standard WHO protocol. The questionnaire included medical history, reproductive history, current smoking/alcohol habit, and work history. Smokers and alcohol consumers were excluded from the study to avoid errors. The objective of the research work was explained to the participants, and a written consent form was taken from the participants before sample collection.

Methodology

Collection of sample

Samples were collected with the help of a lab technician under supervision. As soon as samples were collected, they were transported to the lab for further processing. Before semen collection, the participants were advised to abstain for 3-4 days. One part of the semen was stored at −20°C in a lead-free storage vial for measurement of seminal fluid lead content. For the estimation of blood lead (B-lead) levels, 2 mL of morning, fasting venous blood was collected in heparin vials.

Semen analysis

Semen was analyzed by following a WHO standard protocol, where the volume, pH, viscosity, liquefaction, sperm motility, total sperm count, and sperm morphology were estimated.

Blood-lead estimation

Venous blood (total 2 mL) was sampled into heparin-containing vials for the analysis of B-lead levels. The method has been performed by following the standard Flame Atomic Absorption Method (NIOSH Manual of Analytical Methods, 4th edition) (Flame AAS, Model-SL168; ELICO).

Sperm DNA integrity by the comet assay

The sperm DNA integrity was determined by modified alkaline single-cell gel electrophoresis and the comet assay method described by McKelvey-Martin et al. (1997); further, the protocol was modified using the silver stain technique (Ahuja and Saran, 1999), which is easily accessible in the laboratory. After electrophoresis, the slides were processed with the silver-staining solution (two washes, 10 min each). Coded slides were viewed using a bright-field microscope (Leica microscope). For each sample, 100 randomly selected sperm nuclei were evaluated by an image analysis system using software attached to the computer provided by the Leica microscope, and the tail length was measured. The formula is as follows: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*} {\rm Sperm \ DNA \ tail \ length \ (\mu m)}={\rm (Length \ of \ the \ comet \ sperm \ cell) }\\ \quad- \ {\rm (Breadth \ of \ the \ comet \ sperm \ cell)} \end{align*} \end{document}

Results

The results of B-lead levels are depicted in Table 1. The data reveal that the mean B-lead levels were higher in group I, followed by group II and group III. Macroscopic semen parameter variables in the study group are given in Table 2. The data show that there is no significant alteration in the volume, pH, and liquefaction time among the control and exposed groups. The data on conventional microscopic semen analysis are presented in Table 3, and there is a statistically significant decrease in the percentage of sperm motility (χ²=27.93, p<0.001) and total sperm count (χ²=28.01, p<0.001), and also, there is a significant increase in abnormal sperm morphology in the exposed groups compared with the age-matched control group (χ²=50.77, p<0.001) before vitamin C prophylaxis. Results are shown in Table 4. There is no significant deviation in macroscopic semen parameters after vitamin C prophylaxis. The industrial workers showed a statistically significant increase in sperm motility (t=−5.5, p<0.001) and the sperm total count (t=−4.9, p<0.001) and a statistically significant decrease in abnormal sperm morphology (t=−4.2, p<0.001) after vitamin C prophylaxis. The comet assay also shows similar results, where there is a statistically significant decrease in alkaline-labile sites and a statistically significant decrease in the mean tail length of the comet when compared to the control group (t=2.48, p<0.001) after vitamin C prophylaxis (Tables 5 and 6).

Table 1.

Blood-Lead Levels in the Study Population

	Controls (120)	Group I (n=40)	Group II (n=40)	Group III (n=40)
Blood-lead levels (μg/dL)	5.1±1.24	77.17±7.89	63.58±4.32	56.12±3.24

Table 2.

Macroscopic Semen Parameters of the Study Group

Semen parameters	Control group (n=120)	Infertile group	t-Test values
Semen volume (mL)	2.45±1.11	Group I (n=40)	1.836^a
		2.08±0.59
		Group II (n=40)	1.376^a
		2.06±0.62
		Group III (n=40)	1.001^a
		2.01±0.45
Semen pH	7.45±0.18	Group I (n=40)	3.150^a
		7.34±0.92
		Group II (n=40)	2.516^a
		7.34±0.05
		Group III (n=40)	1.505^a
		7.38±0.04
Semen liquefaction (min)	29.1±6.74	Group I (n=40)	−1.909^a
		31.6±5.93
		Group II (n=40)	−1.963^a
		32.24±4.01
		Group III (n=40)	−1.444^a
		32.24±4.32

Values are mean±SE.

Not significant.

SE, standard error.

Table 3.

Microscopic Semen Parameters of the Study Group

Semen parameters	Control group (n=120)	Infertile group	t-Test values
Total sperm count (×10⁶ spermatozoa/mL)	58.91±9.66	Group I (n=40)	18.066^a
		23.85±8.46
		Group II (n=40)	14.753^a
		23.20±8.60
		Group III (n=40)	10.434^a
		32.24±4.32
Sperm motility (%)	72.0571±1.08555	Group I (n=40)	21.751^a
		30.9714±1.55649
		Group II (n=40)	18.354^a
		30.0500±1.97281
		Group III (n=40)	12.454^a
		42.0000±1.15470
Normal sperm morphology (%)	72.05±9.01	Group I (n=40)	21.739^a
		30.97±9.07
		Group II (n=40)	15.315^a
		30.05±8.59
		Group III (n=40)	9.321^a
		40.32±4.32
Abnormal sperm morphology (%)	23.77±7.60	Group I (n=40)	−27.086^a
		65.7±11.2
		Group II (n=40)	−19.106^a
		49.43±4.30
		Group III (n=40)	−13.252^a
		51.0±4.30

p<0.001.

Table 4.

Effect of Vitamin C Prophylaxis in the Study Population

	Volume	pH	Liquefaction	Total count	Motility	Abnormal
Controls (n=120)	2.45±1.11	7.45±0.18	29.1±6.74	58.91±9.66	72.05±9.01	23.77±7.6
After vit. C prophylaxis	NA	NA	NA	NA	NA	NA
Group I (n=40)	2.08±0.59	7.34±0.92	31.6±5.93	23.85±8.46	30.97±9.07	65.7±11.2
After vit. C prophylaxis	2.06±0.62	7.21±0.81	30.11±6.1	34.24±8.3^a	41.08±9.02^a	59.37±5.4^b
Group II (n=40)	2.01±0.45	7.34±0.05	32.24±4.0	23.2±8.6	30.05±8.59	49.43±4.3
After vit. C prophylaxis	2.02±0.4	7.34±0.02	31.12±4.2	33.95±9.31^b	40.6±8.83^a	41.16±5.2^a
Group III (n=40)	2.16±0.37	7.38±0.04	32.24±4.3	32±4.32	40.32±4.32	51±4.3
After vit. C prophylaxis	2.2±0.29	7.27±0.02	31.14±4.45	42±4.32^a	52±4.35^a	45.66±3.65^a

Values are mean±SE.

p<0.0001.

p<0.001.

vit. C, vitamin C; NA, not applicable.

Table 5.

Sperm Comet Tail Length of the Study Group

	Control group (n=120)	Infertile group	t-Test values
Sperm comet tail length (μm)	1.92±0.32	Group I (n=40)	−12.525^a
		5.4649±2.31
		Group II (n=40)	−9.063^a
		5.1500±2.3
		Group III (n=40)	−11.022^a
		4.5220±1.86

Values are mean±SE.

p<0.001.

Table 6.

Effect of Vitamin C Prophylaxis on DNA Integrity in Study Population

	Controls (n=120)	Group I (n=40)	Group II (n=40)	Group III (n=40)
Comet tail length	1.92±0.32	5.46±2.31	5.15±2.3	4.5±1.86
After vit. C prophylaxis	1.94±0.33	4.82±1.69^a	4.28±1.91^a	3.51±1.36^b

Values are mean±SE.

p<0.001.

Discussion

Lead poisoning was among the first known and most widely studied work and environmental hazards (Flora et al., 2008). Because lead has been used widely for centuries, the effects of exposure are worldwide. Environmental lead is ubiquitous, and everyone has some measurable B-lead level (Hu et al., 2007; Karri et al., 2008). Lead is one of the largest environmental medicinal problems in terms of numbers of people exposed and the public health toll it takes. Although regulations reducing lead in products have greatly reduced exposure in the developed world since the 1970s, lead is still allowed in products in many developing countries (Pokras and Kneeland, 2008). In all countries that have banned leaded gasoline, average B-lead levels have fallen sharply (Meyer et al., 2008). However, some developing countries still allow leaded gasoline (Payne, 2008), which is the primary source of lead exposure in most developing countries. Beyond exposure from gasoline, the frequent use of pesticides in developing countries adds a risk of lead exposure and subsequent poisoning (Konradsen and Cole, 2003).

The Centers for Disease Control have set the standard elevated B-lead level for adults to be 25 (μg/dL) of the whole blood. For children, however, the number is set much lower at 10 (μg/dL) of blood, and in 2012, there were recommendations to reduce this to 5 (μg/dL) (Grant, 2009; Centers for Disease Control and Prevention, 2012). From our results tabulated in Table 1, it was clear that group I (assembly unit) workers have B-lead levels above the biological exposure index limit of 30 μg/dL, as suggested by ACGIH (2002), when compared to group II (plate-buffing unit) and group III (dry charging unit) workers. This suggests that in these units, that is, assembly unit and plate-buffing unit, lead is handled directly when compared to the dry-charging unit, and the same has been suggested by Ravichandran et al. (2005).

In Table 3, group I shows the maximum percentage of abnormal sperm morphology, when compared to group II and group III, showing high susceptibility to lead toxicity. Likewise, total sperm count data and sperm motility data of group I are less than that of group II and group III. From the above investigation, we can conclude that lead interferes with testicular function and also exerts its effects on the sperm DNA, leading to fragmentation. Similar results were reported on studies conducted on European workers occupationally exposed to lead (Tong et al., 2000). A study conducted on toll-gate workers who were exposed to traffic pollution (lead as the main component) also showed a reduction in semen parameters.

In Table 4, data on vitamin C prophylaxis show that there is a significant increase in sperm motility and sperm count, as well as decrease in abnormal sperm morphology after administration of vitamin C. The present data show a significant reduction in the incidence of sperm DNA fragmentation in ejaculated spermatozoa after 3 months of oral antioxidant treatment (Tables 5 and 6). Antioxidant therapy will offer a commendable protection against ROS-induced male infertility by oral treatment with two antioxidants, vitamin C and E (Greco et al., 2005). A combination of vitamin E and C administered orally for 2 months resulted in improvement of the sperm concentration (Kodama et al., 1997). Hence, vitamin C can improve the quality of semen by protecting it from oxidative stress. Vitamin C acts as a major chain-breaking antioxidant and protects sperm from oxidative damage (Wang et al., 2002). The use of vitamin C in several clinical trials has been rather controversial (Rolf et al., 1999). To the contrary, some studies have found no changes in sperm characteristics after treatment with vitamin C or E. From the results, our studies show the protective role of vitamin C against lead-induced oxidative stress in spermatozoa. A few authors also reported that vitamin C enhances sperm quality, protecting sperm and the DNA from the damage caused by oxidative stress, and even basic semen parameters have shown to be improved by oral antioxidant treatment (Martin and Sakkas, 1998). Studies on smokers showed an improvement in the semen quality after vitamin C prophylaxis of 1000 mg/day (Dawson et al., 1991). With a pharmacological supplementation of vitamin C (1 g/day), a more than twofold increase in the plasma ascorbic acid concentration can be achieved (Wen et al., 1997). Furthermore, vitamin C protects human spermatozoa against endogenous oxidative DNA damage (Fraga et al., 1991). Ascorbic acid was shown to exert its protective role against the sperm DNA damage in 75 infertile men (which will support the present observation) (Song et al., 2006). Hence, further investigations are required to document the significant role played by the antioxidant-enriched compounds by targeting a large number of human subjects and by considering several other confounding factors.

Conclusions and suggestions to mitigate the problem

It is thus concluded from our study that supplementation of vitamin C to exposed workers reduces the risk of male infertility. However, the present work is a preliminary approach toward an antioxidant therapy in reducing the degree of male infertility. Further investigation has to be made by targeting larger populations over a period and also by using a mixture of antioxidants in the therapy.

Reproductive epidemiological studies are needed to be encouraged so as to minimize the effects of chemicals/pollutants on the human body. Research in this particular area is required to be encouraged by funding agencies and institutions so as to understand the interaction of chemicals/pollutants with the reproductive system. Further research is required in this field to fully understand the complicated events associated with oxidative stress and to eventually develop effective strategies to assist patients with oxidative stress-associated male infertility. In conclusion, reproductive epidemiological studies by targeting a large number of samples with several confounding factors are to be encouraged to assess the effect of environmental pollution on the health of the individuals.

Footnotes

Acknowledgments

We are thankful to the Central Research Institute of Unani Medicine, Hyderabad, and Chair for Medicine and Molecular Genetics, College of Applied Medical Sciences, King Saud University, for providing fund and support in completing the project.

Author Disclosure Statement

No competing interests exist.

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