Abstract

Epidemiological studies finding menstrual cycle abnormalities among women occupationally exposed to Hg° prompted us to investigate the mechanisms of reproductive toxicity of Hg° in the female rat. Nose-only Hg° vapor inhalation exposures were conducted on regularly cycling rats 80–90 days of age in dose-response and acute time-course studies, which have previously proven useful as a model to identify ovarian toxicants. Vaginal smears were evaluated daily and serum hormone levels were correlated with cycle and with ovarian morphology at necropsy. Exposure concentration–related effects of Hg° were evaluated by exposing rats to 0, 1, 2, or 4 mg/m3 Hg° vapor 2 h/day for 11 consecutive days. Tissue Hg levels correlated with exposure concentration and duration. Exposure of rats to 4 mg/m3 (but not 1 or 2 mg/m3) Hg vapor for 11 days resulted in significant decreases in body weights relative to controls. Estrous cycles were slightly prolonged in the 2 and 4 mg/m3 dose groups, and serum estradiol and progesterone levels were significantly different in the 4 mg/m3 group compared to controls. The alterations in cycle and hormones at the 4 mg/m3 exposure concentration were attributed to body weight loss and generalized toxicity. In the time-course study, rats were exposed to 2 mg/m3 Hg° or air beginning in metestrus and evaluated daily for 8 days. A lengthening of the cycle was detected and morphological changes were observed in the corpora lutea (CL) after exposure for 6 days. To determine if changes in the CL and cyclicity correlated with a functional defect, rats were exposed to Hg° vapor and evaluated for pregnancy outcome. There were no significant effects on pregnancy rate or numbers of implantation sites when rats were exposed to 1 or 2 mg/m3 Hg° for 8 days prior to breeding, or when exposed for 8 days after breeding. These studies indicate that exposure to Hg° vapor altered estrous cyclicity, but had no significant effect on ovulation, implantation, or maintenance of first pregnancy during exposure of short duration in female rats.

Elemental (metallic) mercury (Hg°) is a highly hazardous chemical that can cause serious adverse health effects. Elemental Hg° is the liquid form of mercury found in thermometers, fluorescent light bulbs, barometers, blood pressure instruments, mercury switches in children's shoes that light up, and in dental amalgams (ATSDR, 1997). Elemental Hg° is poorly absorbed by the dermal and oral routes. However, because it is highly volatile, the primary route of human exposure is by inhalation of Hg° vapors. Inhaled Hg° vapor easily crosses the pulmonary capillary membranes and can accumulate in distal tissues (Magos, 1967).

The tissue distribution of Hg is similar after inhalation of Hg° vapors or after ingestion of inorganic mercuric salts (Hayes and Rothstein, 1962; Rothstein and Hayes, 1960). However, because Hg° more readily penetrates cellular membranes than inorganic mercuric salts, inhalation of Hg° vapors results in greater Hg accumulation in all tissues. Intracellular Hg° is rapidly oxidized by cytosolic catalase to mercuric mercury (Hg2+), the reactive species for most Hg compounds. Hg2+ can be formed by oxidation of Hg°, reduction of mercuric salts, or demethylation of methylmercury (IPCS, 1991). Because Hg2+ is highly reactive, it rapidly combines with intracellular ligands such as sulfhydryls, potentially disrupting enzymes and proteins essential to normal organ function.

Although data on the reproductive toxicity of Hg° are limited, epidemiological studies have documented menstrual cycle abnormalities among women exposed to Hg° vapor. Six studies, conducted mostly in Europe, have reported menstrual cycle abnormalities (including changes in bleeding patterns and cycle length) among women occupationally exposed to Hg° vapor (De Rosis et al., 1985; Goncharuk, 1977; Marinova et al., 1973; Mikhailova et al., 1971; Panova and Dimitrov, 1974; Sikorski et al., 1987). In a more recent study, Rowland et al. (1994) found decreased fertility among female dental assistants exposed to Hg° vapor. Women that prepared more than 30 amalgams per week and who had poor occupational hygiene factors were only 63% as likely as unexposed women to conceive in any given menstrual cycle, whereas women who prepared fewer than 30 amalgams per week or women who prepared more than 30 amalgams per week with the best hygiene factors had better fecundability then even the unexposed women. These observations suggest the need to understand specific exposures of women and to understand how Hg may affect the reproductive system.

Although the mechanism is not clear, all chemical forms of Hg administered to animals have been shown to result in reproductive problems such as spontaneous abortion, stillbirths, congenital malformations, infertility, disturbances in the menstrual cycle, and inhibition of ovulation (for review: Barlow and Sullivan 1982; Schuurs, 1999). Most studies have been conducted with the inorganic forms of Hg and with systemic toxicity and lethality often a reported outcome. The one study of Hg° vapor that we are aware of reported that whole-body exposure to 2.5 mg/m3 Hg° for 6 h daily for 6–8 weeks caused a lengthening of the estrous cycle and postnatal pup mortality in treated female rats (Baranski and Szymczyk, 1973). However, specific mechanisms of this reproductive toxicity were not identified. Moreover, 6 of 24 exposed rats died due to Hg poisoning, suggesting that the rats were systemically sick, which in turn may have secondarily suppressed reproductive activity including estrous cyclicity. Our studies described herein investigate whether exposure to Hg° vapor at concentrations that do not cause systemic toxicity can change endocrine profiles over time and perturb the female reproductive system.

MATERIALS AND METHODS

Mercury Vapor (Hg°) Exposures

Generation and monitoring.

Hg° vapor was generated by passing conditioned air (HEPA filtered, charcoal-scrubbed, temperature and humidity controlled) through a flask containing elemental Hg (Aldrich Chemical Co., Milwaukee, WI). The resulting Hg° vapor was diluted and delivered to the exposure system at a controlled rate using mass flow controllers. Two nose-only exposure systems (Lab Products, Rockville, MD) were used, one for exposing control animals to conditioned air, and the other for exposing animals to Hg° vapor. Each experiment consisted of one Hg° concentration group and a concurrent air-exposed control group. Air samples from the exposure system were analyzed every 15 min using a Jerome Model 431-X Mercury Analyzer (Arizona Instruments Phoenix, AZ).

Animals.

Female Sprague-Dawley rats (61–67 days of age; Charles River Breeding Laboratories, Raleigh, NC) were housed three per cage. Feed (NIH-07) and water (deionized, filtered tap water) were provided ad libitum except during the 2-h exposures. During the 2 weeks prior to exposure, rats were acclimated to the exposure schedule by removing food and water each morning for 2 h. Body weights were recorded daily.

Estrous cyclicity.

Vaginal cytology was determined daily for each animal beginning 9 days before exposure and up to 11 days during Hg° exposure. The vaginal vaults were lavaged with sterile saline pH 7.4, and the aspirated lavage fluid and cells were stained with Wright's stain. Relative numbers of leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain the estrous cycle stage (i.e., diestrus, proestrus, estrus, or metestrus). Cycle length was characterized as 4-day, 5-day, or greater than 5-day cycles. Rats with two consecutive 4-day cycles were selected for study and then randomized by weight to treatment groups.

Animal exposures.

Animals were placed in cylindrical holding tubes during nose-only exposures. Preliminary experiments demonstrated that confinement of rats in holding tubes for longer than 2 h per day for up to 10 days (air exposure) resulted in significant reductions in body weight gain; for this reason, the exposures were limited to 2 h. In the dose-response study, rats were exposed for 11 days to 1 mg/m3 Hg° (n = 9), 2 mg/m3 Hg° (n = 15), or 4 mg/m3 Hg° (n = 18), or conditioned air as controls (n = 9, 15, or 18 per comparison group, respectively). In the time-course study, rats (n = 6 per time point per dose for a total of 48 control and 48 treated at 2 mg/m3 Hg° ) were exposed for 8 consecutive days beginning on metestrus or diestrus of the cycle. The functional breeding studies were separated into two study designs, referred to as the Expose-Mate Study and the Mate-Expose Study. In the Expose-Mate study, rats (n = 6–12) were exposed to air or 2 mg/m3 Hg° for a minimum of 8 days and then a control and Hg°-treated rat were paired with a male overnight as exposure continued during the day. After sperm-positive smears were observed, exposure was discontinued and female rats were placed in holding cages until necropsied 7 days after mating. The Mate-Expose study was designed to distinguish effects on early implantation loss. Female rats were bred (n = 6 per dose group) and then exposed to 0, 1, or 2 mg/m3 Hg° for 8 days before necropsy. At necropsy, implantation sites and number of CL were determined.

Serum hormone analyses.

Rats were anesthetized by CO2 inhalation and blood was collected by cardiac puncture. Serum was collected and stored at –70° until analyzed for hormones. Estradiol and progesterone were measured by radioimmunoassay using commercially available kits from Diagnostic Products, Inc. (Los Angeles, CA).

Histopathology.

Lungs, liver, right kidney, ovary, and uterus were collected from treated and control rats at necropsy. Tissues were trimmed and fixed in 10% neutral-buffered formalin. Paraffin-embedded sections were stained with hematoxylin and eosin and examined by light microscopy.

Urine and tissue Hg analyses.

Urine was collected daily during each 2-h exposure. Individual animal urine volumes were recorded and then urine from all exposed rats was pooled. Immediately after the last exposure, all rats were euthanized by CO2:O2 anesthesia followed by exsanguination. Brain, kidney, liver, ovary and uterus were collected in acid-washed glass vials (prepared in a class 100 clean room) and stored at –20°C until analyzed. Tissue samples were weighed, homogenized, and digested overnight at 70°C in sealed vials. After neutralization and dilution, samples were analyzed for total Hg by cold vapor atomic fluorescence spectrometry (Stockwell and Corns, 1993).

Statistical analyses.

Student's t-test was used to determine statistically significant differences between body weights, organ weights, hormone levels, and tissue Hg levels in treated and control animals. Chi square analysis was used to determine estrous cycle differences (Sokal and Rohlf, 1969).

RESULTS

Dose-Response Study

Body and organ weights.

Exposure of rats to 4 mg/m3 Hg° vapor for 7 and 11 days resulted in significant decreases in body weights relative to controls (Table 1). Body weights were not affected at exposure concentrations of 1 or 2 mg/m3. Absolute and relative brain, kidney, liver, and uterus weights were not significantly changed by exposure to 1, 2, or 4 mg/m3 Hg° for 11 days (data not shown).

Urinary Hg.

Total urinary Hg levels increased with increasing exposure concentration and duration (Table 2). Urinary Hg levels increased proportionately at the lower exposure concentrations. When examined at day 11, the urinary Hg levels in the 2 mg/m3 group (53 ng/g) were about 2-fold greater than in the 1 mg/m3 concentration group (19.1 ng/g). However, urinary Hg levels in the 4 mg/m3 group (842 ng/g) were about 16-fold greater than in the 2 mg/m3 group. Total Hg levels in urine of control rats were very low (0.44 ng/g).

Kidney and brain Hg.

After exposure of rats for 11 days, kidney and brain Hg levels increased proportionately with Hg° vapor exposure concentration (Table 3). Doubling the exposure concentration from 1 to 2 mg/m3 and from 2 to 4 mg/m3 resulted in 3-fold and 1.5-fold increases in kidney Hg levels, respectively. In the brain, doubling the exposure concentration from 1 to 2 mg/m3 resulted in a 2.3-fold increase, and from 2 to 4 mg/m3 resulted in a 3.8-fold increase in Hg levels. Hg levels in kidney were 20- to 60-fold higher than levels measured in brain.

Vaginal cytology.

Rats exposed to 2 and 4 mg/m3 Hg° vapor for 11 days had a greater percentage of estrous cycles that were 5 days, or greater than 5 days (no cycle) compared to controls (Table 4). Exposure to 1 mg/m3 Hg° vapor had no significant effect on the estrous cycle.

Serum hormones.

In rats exposed to 4 mg/m3 Hg° vapor, estradiol was decreased (17 ± 2.1 pg/ml) compared to controls (33 ± 9 pg/ml), and progesterone was increased (14 ± 2.5 ng/ml) relative to controls (5.86 ± 1.2 ng/ml). No significant changes in serum hormones were found in the other exposure groups relative to controls (data not shown).

Time Course Study

Organ weights.

Exposure of rats to 2.0 mg/m3 Hg° resulted in a significant decrease in relative liver weights after exposure for 4 to 8 days; however, the biological significance of this change is unclear. Brain, kidney, lung, and uterus weights were unchanged relative to untreated controls (data not shown).

Ovarian histopathology.

Morphological changes were observed in the corpora lutea of Hg-exposed rats that were necropsied at estrus or metestrus. The corpora lutea of exposed rats appeared immature compared to controls. Although morphologically different, the number of corpora lutea were similar between exposed and control rats, and ovulation was confirmed in both groups by the presence of oocytes in the oviducts.

Vaginal cytology.

The estrous cycles of exposed rats were prolonged after exposure for 6 to 8 days to 2 mg/m3 Hg° vapor. The morphological changes in the corpora lutea (described above) were observed at this time.

Ovary Hg content.

Ovaries were analyzed for total Hg content after exposure of rats to air or 2 mg/m3 Hg° vapor for up to 8 days (Table 5). Total Hg levels in ovaries of exposed rats were significantly increased over controls after the initial exposure, and continued to increase during the 8-day study. Total Hg levels in control ovaries were very low (0.003 μg/g).

Serum hormones.

Exposure to 2 mg/m3 Hg° vapor for 8 days had no significant effect on serum levels of estrogen and progesterone as shown by estrogen:progesterone ratios (Table 5).

Expose-Mate Study

Relative to controls, there were no significant effects on mating efficiency, numbers of sperm positive females, numbers of corpora lutea and implantation sites, or hormone levels in female rats exposed to 2 mg/m3 Hg° vapor for 8 days prior to housing with males (Table 6).

Mate-Expose Study

Relative to controls, there were no significant effects on numbers of corpora lutea or implantation sites, or on hormone levels in female rats that were first bred and then exposed to 2 mg/m3 Hg° vapor for 8 days (Table 7).

DISCUSSION

Exposure to relatively high Hg° vapor concentrations has been reported to cause reproductive dysfunction in women and in laboratory animals (Schuurs, 1999); however, the mechanism(s) by which Hg° causes reproductive toxicity has not been adequately addressed. In laboratory animal studies, exposure to relatively high concentrations of Hg° results in chemical-related effects such as weight loss or other secondary toxic effects known to confound interpretation of reproductive studies. A rat model of reproductive toxicity was used in these studies to investigate whether inhalation exposure to Hg° vapor could perturb the female reproductive system at exposure concentrations that did not cause systemic toxicity.

In addition to avoiding acute chemical-related effects, it was also essential to minimize unnecessary stress to the rats caused by confinement during nose-only exposure. Preliminary studies with this rodent model established that nose-only exposure for greater than 2 h per day, even after acclimation, reduced body weight gain and altered estrous cyclicity in female rats (data not presented). Consequently, rats were only exposed for 2 h/day, which ultimately limited the dose of Hg but allowed the distinction between reproductive toxicity and systemic toxicity.

Approximately 80% of inhaled Hg° is absorbed from the alveoli and distributed throughout the body (IPCS, 1991). In order to monitor the tissue dose of Hg under our exposure regimen, urine, kidney, and brain Hg levels were measured. Even though the exposure duration was limited to 2 h/day, relatively high levels of Hg were attained in brain, kidney, and urine. The kidney is the primary depository for Hg (IPCS, 1991), and in this study Hg concentrations in kidney were 20- to 60-fold higher than those measured in the brain. Because nephrotoxicity is an early symptom of Hg poisoning, the kidney was evaluated microscopically for evidence of tissue injury. However, even at the high tissue Hg levels attained in this study, there was no histological evidence of toxicity in the kidney. Doubling the Hg° exposure concentration (1, 2, or 4 mg/m3) resulted in proportionate increases in kidney and brain Hg levels. However, the amount of Hg excreted in urine increased approximately 16-fold when the exposure concentration was doubled from 2 to 4 mg/m3. This nonlinear increase in urinary Hg levels indicates a potential saturation of Hg uptake and storage at the 4 mg/m3 concentration level.

The dose-response studies indicated that exposure to 4 mg/m3 Hg° for 2 h/day was too toxic, causing significant body weight loss after only 7 days of exposure. In addition, the nonlinear increase in urinary Hg output indicated that tissue uptake was saturated at this exposure concentration. For these reasons, the significant changes in estrous cycle and serum hormones observed at this high exposure concentration were attributed to systemic toxicity and not due to a specific effect of Hg° on the reproductive system. However, alterations in estrous cyclicity without significant weight loss were observed in the 2 mg/m3 Hg° vapor exposure group. Therefore, this exposure concentration was chosen to investigate the potential reproductive toxicity of Hg° vapor in a more sensitive time-course study.

The time-course study similarly indicated that exposure to 2 mg/m3 Hg° slightly prolonged the estrous cycle and altered the morphology of the corpora lutea; however, there were no concomitant changes in serum estradiol or progesterone levels. Prolongation of the estrous cycle (Lamperti and Printz, 1974) and morphological changes in corpora lutea with inhibition of follicular maturation (Lamperti and Printz, 1973) have been observed after injection of HgCl2 into female hamsters. Although estradiol and progesterone were not measured in those studies, follicle-stimulating hormone in the pituitary was reportedly elevated (Lamperti and Niewenhuis, 1976).

Because the corpora lutea of cycling animals are not functional unless mating occurs, it was possible that Hg° could interfere with the ability of the corpora lutea to maintain progesterone secretion and thereby maintain pregnancy, but this adverse effect would not be detected in a cycling animal. For this reason, it was difficult to interpret the functional significance of the subtle morphological changes in corpora lutea. Two possibilities were that the corpora lutea were altered due to Hg° vapor exposure, or that the changes were secondary and related to the time in the cycle that these rats were evaluated. Therefore, we reasoned that evaluation of pregnancies would assess the functionality of the corpora lutea and also test the integrity of the hypothalamus-pituitary-ovarian-uterine system. Two approaches were adopted. The first was to expose females to Hg° and then assess ovulation and mating efficiency (the number of mated females that became pregnant) by measuring sperm positivity, the number of CL (representing ovulations), and the number of implantations. If implantations were decreased, it would still be possible that Hg° affected the ovary and/or the pup. Therefore, the second study design was to mate the female, then expose to Hg° vapor to assess early implantation loss due to fetotoxicity separate from ovarian function (Cummings, 1993).

Under these conditions, no differences in mating efficiency, implantation numbers, or numbers of corpora lutea were found between control and Hg°-exposed rats. Thus, we conclude that Hg° had no measurable effect on corpora luteal function in this exposure scenario. Baranski and Szymczyk (1973) reported a lengthening of the estrous cycle in Hg° vapor-exposed rats with no effect on mating efficiency; however, they observed a significant reduction in the number of implantations. The reduced number of implantations was likely an indirect effect of Hg° toxicity, as the exposure duration was considerably longer in the Baranski and Szymczyk study (2.5 mg/m3 for 6 h/day, 5 days/week for 21 days), and rats developed symptoms of Hg poisoning (hyperactivity, chronic seizures, and whole body trembling) in the second and third weeks of exposure.

The subtle changes in estrous cyclicity and corpora luteal morphology observed in the current study may indicate slight perturbations in the feedback systems between the hypothalamus, pituitary, and ovary, as these alterations do not affect steroid hormone levels or functional fertility. Hg has been shown to accumulate in the hypothalamus (Ernst et al., 1993) and pituitary (Kosta et al., 1975) of rats exposed to Hg° vapor, and in the current study, high levels of Hg accumulated in the ovaries of Hg°-exposed rats. The reaction of Hg with essential intracellular proteins in these tissues could disrupt key feedback systems involved in maintaining the timing or biological clocks associated with estrous cyclicity.

These Hg° vapor inhalation studies were designed to investigate the mechanisms of Hg° vapor toxicity in the female rat to support the epidemiological findings and to aid in the determination of public health risks. Our results demonstrate that Hg° vapor inhalation causes systemic toxicity, but does not cause ovarian or reproductive dysfunction within a short exposure period. It is possible that exposure to lower concentrations for longer exposure periods (weeks, months, or years) would demonstrate a direct reproductive effect of Hg° vapor. This speculation is suggested by the finding that Hg° vapor exposure caused a slight estrous cycle lengthening in these short-term studies, and estrous cycle length is a useful marker of overall reproductive function in evaluating reproductive toxicants in a continuous dosing and continuous breeding protocol (Chapin et al., 1997). However, long-term studies with Hg° are not feasible, given its highly toxic nature and the necessity to use nose-only exposures in our animal model. Because of the complications and limitations of dosing, it is equally difficult to directly compare exposure levels in our acute studies in rats and exposure levels in occupationally exposed women. Women who exhibit apparent reproductive problems due to occupational exposures have poor hygiene practices and/or exceed the time-weighted long-term threshold limit value of 10 μg/m3 Hg° in air (Schuurs, 1999). Given that Hg is a highly hazardous chemical, we suggest that reproductive effects noted in occupationally exposed women might be secondary to systemic and/or neurotoxic effects of Hg, or perhaps symptomatic of exposures to multiple chemicals in the workplace and the environment.

TABLE 1

Body Weights of Female Rats Exposed to Hg Vapor

 Number of exposures  
Hg° exposure concentration 11  
Note. Rats were exposed to 4 mg/m3 Hg vapor 2 h/day for 7 and 11 days. Values represent means ± SD (n). 
aBody weights in grams. 
bBody weight treated (g)/body weight of control (g) × 100. 
*Significantly less than controls (p < 0.05). 
1 mg/m3 250    ±    11     (9)a 251    ±    11     (9)  257    ±    12     (9) 257    ±    13     (9)  
Control 258    ±    24     (8) 258    ±    25     (8)  264    ±    28     (8) 267    ±    31     (8)  
% Controlb  97  97  97  96  
2 mg/m3 252    ±    20     (14) 248    ±    20     (14) 250    ±    22     (14) 251    ±    2     (14)  
Control 246    ±    22     (13) 241    ±    23     (13) 245    ±    24     (13) 251    ±    24     (12)  
% Control 102 103 102 100  
4 mg/m3 243    ±    18     (18) 236    ±    17     (18) 227    ±    17     (18)* 219    ±    19     (18)*  
Control 244    ±    19     (18) 245    ±    20     (18) 246    ±    20     (18) 248    ±    21     (18)  
% Control  99  96  92  88 
 Number of exposures  
Hg° exposure concentration 11  
Note. Rats were exposed to 4 mg/m3 Hg vapor 2 h/day for 7 and 11 days. Values represent means ± SD (n). 
aBody weights in grams. 
bBody weight treated (g)/body weight of control (g) × 100. 
*Significantly less than controls (p < 0.05). 
1 mg/m3 250    ±    11     (9)a 251    ±    11     (9)  257    ±    12     (9) 257    ±    13     (9)  
Control 258    ±    24     (8) 258    ±    25     (8)  264    ±    28     (8) 267    ±    31     (8)  
% Controlb  97  97  97  96  
2 mg/m3 252    ±    20     (14) 248    ±    20     (14) 250    ±    22     (14) 251    ±    2     (14)  
Control 246    ±    22     (13) 241    ±    23     (13) 245    ±    24     (13) 251    ±    24     (12)  
% Control 102 103 102 100  
4 mg/m3 243    ±    18     (18) 236    ±    17     (18) 227    ±    17     (18)* 219    ±    19     (18)*  
Control 244    ±    19     (18) 245    ±    20     (18) 246    ±    20     (18) 248    ±    21     (18)  
% Control  99  96  92  88 
TABLE 2

Total Hg Concentration in Urinea

 Number of exposures  
Hg° exposure concentration 11  
Note. Urine was collected from rats immediately after the 2-h exposure to Hg° vapor or air (control). Urine from five rats/group was pooled and analyzed for total Hg. The average Hg concentration in control urine was 0.44 ng/gm. 
aTotal Hg concentration in nanograms per gram urine. 
1 mg/m3 3.2 8.6 9.5 11.8 12.7 19.1  
2 mg/m3 12.1 20.1 38.0 43.4 48.7 52.7  
4 mg/m3 41.0 112.3 144.6 583.4 613.7 841.6 
 Number of exposures  
Hg° exposure concentration 11  
Note. Urine was collected from rats immediately after the 2-h exposure to Hg° vapor or air (control). Urine from five rats/group was pooled and analyzed for total Hg. The average Hg concentration in control urine was 0.44 ng/gm. 
aTotal Hg concentration in nanograms per gram urine. 
1 mg/m3 3.2 8.6 9.5 11.8 12.7 19.1  
2 mg/m3 12.1 20.1 38.0 43.4 48.7 52.7  
4 mg/m3 41.0 112.3 144.6 583.4 613.7 841.6 
TABLE 3

Total Hg in Kidney and Brain of Rats Exposed to Hg° Vapor

Hg° exposure concentration Kidney (μg/g) Brain (μg/g)  
Note. Rats were exposed to Hg° vapor 2 h/day for 11 consecutive days. Immediately after the last exposure, rats were euthanized and tissues were collected and analyzed for total Hg. Values represent means ± SD (n). 
Control 0.066    ±    0.019     (7) 0.002    ±    0.002     (6)  
1 mg/m3 32    ±    16     (8) 0.70    ±    0.06     (2)  
2 mg/m3 96    ±    64     (12) 1.6    ±    0.4     (5)  
4 mg/m3 142    ±    40     (12) 6.1    ±    1.0     (5) 
Hg° exposure concentration Kidney (μg/g) Brain (μg/g)  
Note. Rats were exposed to Hg° vapor 2 h/day for 11 consecutive days. Immediately after the last exposure, rats were euthanized and tissues were collected and analyzed for total Hg. Values represent means ± SD (n). 
Control 0.066    ±    0.019     (7) 0.002    ±    0.002     (6)  
1 mg/m3 32    ±    16     (8) 0.70    ±    0.06     (2)  
2 mg/m3 96    ±    64     (12) 1.6    ±    0.4     (5)  
4 mg/m3 142    ±    40     (12) 6.1    ±    1.0     (5) 
TABLE 4

Effect of Hg° Vapor Exposure on Estrous Cyclicity of Rats Exposed for 11 Days

Cycle length Control 1 mg/m3 2 mg/m3 4 mg/m3 
Note. Female rats with two consecutive 4-day cycles were selected for this study. Rats were exposed to Hg° vapor 2 h/day for 11 consecutive days and estrous cyclicity was determined by daily vaginal lavage. Cycle length was determined by the number of days between estrus smears. Data in table given as % rats. % rats determined by number of rats with specified cycle length/number of rats in group × 100. 
*Significantly different from expected controls (p < 0.01). 
4 Days 55 60 25* 11*  
5 Days 45 40 67* 67*  
> 5 Days 8* 22* 
Cycle length Control 1 mg/m3 2 mg/m3 4 mg/m3 
Note. Female rats with two consecutive 4-day cycles were selected for this study. Rats were exposed to Hg° vapor 2 h/day for 11 consecutive days and estrous cyclicity was determined by daily vaginal lavage. Cycle length was determined by the number of days between estrus smears. Data in table given as % rats. % rats determined by number of rats with specified cycle length/number of rats in group × 100. 
*Significantly different from expected controls (p < 0.01). 
4 Days 55 60 25* 11*  
5 Days 45 40 67* 67*  
> 5 Days 8* 22* 
TABLE 5

Estrogen:Progesterone Ratios and Total Hg in Ovaries of Rats Exposed to Hg° Vapor

Days of exposure 1 (M) 2 (D) 3 (P) 4 (E) 5 (M) 6 (D) 7 (P) 8 (E)  
Note. Beginning on metestrus (exposure day 1) all rats were exposed to either 2 mg/m3 Hg° vapor or air 2 h/day for 1 to 8 days. Letters in parentheses indicate expected cycle day (M = metestrus, D = diestrus, P = proestrus, E = estrus). Serum hormone estradiol to progesterone ratios and total Hg in the ovaries (μg/gm tissue) were determined on various days of the estrous cycle. Values represent mean ratio of estradiol/progesterone ± SEM. Values for total Hg in ovaries represent mean ± SEM. The average total Hg in control ovaries = 0.003 ± 0.7 μg/g tissue. 
Hg° exposure concentration  
    Control 3.3    ±    0.6 0.8    ±    0.06 2.8    ±    1.4 3.4    ±    2.0 2.9    ±    0.9 4.3    ±    1.6 2.5    ±    0.5 4.2    ±    2.5  
    2 mg/m3 2.6    ±    0.7 1.2    ±    0.16 1.8    ±    4.1 2.4    ±    0.7 3.0    ±    1.0 2.5    ±    0.4 2.4    ±    0.4 4.8    ±    0.7  
Ovary Hg conc. (μg/g) 1.7    ±    1.4 2.3    ±    0.9 4.2    ±    1.8 8.8    ±    4.7 7.8    ±    1.6 7.8    ±    2.7 12.0    ±    4.5 11.4    ±    3.2 
Days of exposure 1 (M) 2 (D) 3 (P) 4 (E) 5 (M) 6 (D) 7 (P) 8 (E)  
Note. Beginning on metestrus (exposure day 1) all rats were exposed to either 2 mg/m3 Hg° vapor or air 2 h/day for 1 to 8 days. Letters in parentheses indicate expected cycle day (M = metestrus, D = diestrus, P = proestrus, E = estrus). Serum hormone estradiol to progesterone ratios and total Hg in the ovaries (μg/gm tissue) were determined on various days of the estrous cycle. Values represent mean ratio of estradiol/progesterone ± SEM. Values for total Hg in ovaries represent mean ± SEM. The average total Hg in control ovaries = 0.003 ± 0.7 μg/g tissue. 
Hg° exposure concentration  
    Control 3.3    ±    0.6 0.8    ±    0.06 2.8    ±    1.4 3.4    ±    2.0 2.9    ±    0.9 4.3    ±    1.6 2.5    ±    0.5 4.2    ±    2.5  
    2 mg/m3 2.6    ±    0.7 1.2    ±    0.16 1.8    ±    4.1 2.4    ±    0.7 3.0    ±    1.0 2.5    ±    0.4 2.4    ±    0.4 4.8    ±    0.7  
Ovary Hg conc. (μg/g) 1.7    ±    1.4 2.3    ±    0.9 4.2    ±    1.8 8.8    ±    4.7 7.8    ±    1.6 7.8    ±    2.7 12.0    ±    4.5 11.4    ±    3.2 
TABLE 6

Expose-Mate Study

 Reproductive parameters  
Hg° exposure concentration Sperm positivea Pregnantb CLc Implantsd P/E ratioe 
Note. Female rats were exposed to air or 2 mg/m3 Hg° 2 h/day for 5 days, and then were housed overnight with males as exposures continued during the day for up to 8 days. After sperm-positive smears were observed, exposure was discontinued. Female rats were necropsied 9 days after mating was confirmed. 
aNumber of sperm-positive females/number of females bred. 
bNumber of pregnant females/number of sperm positive females. 
cCL = number of corpora lutea. Values represent means ± SEM. 
dNumber of implantation sites. Values represent means ± SEM. 
eP/E = progesterone/estrogen. Values represent means ± SEM. 
Control 5/5 4/5 14.0    ±    2 11.5    ±    3.3 7.8    ±    2.5  
2 mg/m3 4/6 3/4 14.8    ±    2 13.6    ±    0.7 4.0    ±    0.3 
 Reproductive parameters  
Hg° exposure concentration Sperm positivea Pregnantb CLc Implantsd P/E ratioe 
Note. Female rats were exposed to air or 2 mg/m3 Hg° 2 h/day for 5 days, and then were housed overnight with males as exposures continued during the day for up to 8 days. After sperm-positive smears were observed, exposure was discontinued. Female rats were necropsied 9 days after mating was confirmed. 
aNumber of sperm-positive females/number of females bred. 
bNumber of pregnant females/number of sperm positive females. 
cCL = number of corpora lutea. Values represent means ± SEM. 
dNumber of implantation sites. Values represent means ± SEM. 
eP/E = progesterone/estrogen. Values represent means ± SEM. 
Control 5/5 4/5 14.0    ±    2 11.5    ±    3.3 7.8    ±    2.5  
2 mg/m3 4/6 3/4 14.8    ±    2 13.6    ±    0.7 4.0    ±    0.3 
TABLE 7

Mate-Expose Study

 Reproductive parameters  
Hg° exposure concentration Sperm positivea Pregnantb CLc Implantsd P/E ratioe 
Note. Female rats were housed overnight with males. After sperm-positive smears were observed, females were exposed to air or 2 mg/m3 Hg° for 8 days and then necropsied. 
aNumber of sperm-positive females/number of females bred. 
bNumber of pregnant females/number of sperm positive females. 
cCL = number of corpora lutea. Values represent means ± SEM. 
dNumber of implantation sites. Values represent means ± SEM. 
eP/E = progesterone/estrogen. Values represent means ± SEM. 
Control 5/5 3/5 16.0    ±    3 6.8    ±    5 4.1    ±    0.8  
2 mg/m3 7/7 4/7 14.7    ±    2 9.6    ±    4 4.7    ±    1.0 
 Reproductive parameters  
Hg° exposure concentration Sperm positivea Pregnantb CLc Implantsd P/E ratioe 
Note. Female rats were housed overnight with males. After sperm-positive smears were observed, females were exposed to air or 2 mg/m3 Hg° for 8 days and then necropsied. 
aNumber of sperm-positive females/number of females bred. 
bNumber of pregnant females/number of sperm positive females. 
cCL = number of corpora lutea. Values represent means ± SEM. 
dNumber of implantation sites. Values represent means ± SEM. 
eP/E = progesterone/estrogen. Values represent means ± SEM. 
Control 5/5 3/5 16.0    ±    3 6.8    ±    5 4.1    ±    0.8  
2 mg/m3 7/7 4/7 14.7    ±    2 9.6    ±    4 4.7    ±    1.0 
1
To whom correspondence should be addressed. Fax (919) 541-7666. E-mail: davis1@niehs.nih.gov.

Inhalation exposures were conducted at the NIEHS inhalation facility under contract to ManTech Environmental Technology, Inc., Research Triangle Park, NC. Tissue Hg analyses were conducted by Research Triangle Institute, Research Triangle Park, NC. The authors acknowledge the technical assistance of C. Colegrove, D. Crawford, P. Dixon, N. Gage, M. Goods, M. Moorman, S. Philpot, P. Rydell, W. Stephens, and T. Ward.

REFERENCES

ATSDR (1997). Agency for Toxic Substances and Disease Registry. National Alert. A warning about continuing patterns of metallic mercury exposure. 06/26/97.
Baranski, B., and Szymczyk, I. (
1973
). Effects of mercury vapors upon reproductive function of the female white rat.
Medycyna Pracy
 
24
,
249
–261.
Barlow, S. M., and Sullivan, F. M. (1982). Reproductive Hazard of Industrial Chemicals. Academic Press, London.
Chapin, R. E., Sloane, R. A., and Haseman, J. K. (
1997
). The relationships among reproductive endpoints in Swiss mice, using the reproductive assessment by continuous breeding database.
Fundam. Appl. Toxicol.
 
38
,
129
–142.
Cummings, A. (1993). Assessment of Implantation in the Rat. In Female Reproductive Toxicology, Methods in Toxicology (J. J. Heindel and R. E. Chapin, Eds.), pp. 194–198. Academic Press, San Diego, CA.
De Rosis, F., Anastasio, S. P., Selvaggi, L., Beltrame, A., and Moriani, G. (
1985
). Female reproductive health in two lamp factories: Effects of exposure to inorganic mercury vapour and stress factors.
Brit. J. Ind. Med.
 
42
,
488
–494.
Ernst, E., Christensen, M. K., and Poulsen, E. H. (
1993
). Mercury in the rat hypothalamic arcuate nucleus and median eminence after mercury vapor exposure.
Exp. Molec. Pathol.
 
58
,
205
–214.
Goncharuk, G. A. (
1977
). Problems relating to the occupational hygiene of women in production of mercury.
Gig. Tr. Prof. Zabol.
 
5
,
17
–20.
Hayes. A. D., and Rothstein, A. (
1962
). The metabolism of inhaled mercury vapor in the rat studied by isotope techniques.
J. Pharmacol. Exp. Ther.
 
138
,
1
–10.
Kosta, L., Byrne, A. R., and Zekenko, V. (
1975
). Correlation between selenium and mercury in man following exposure to inorganic mercury.
Nature
 
254
,
238
–239.
Lamperti, A. A., and Printz, R. H. (
1973
). Effects of mercuric chloride on the reproductive cycle of the female hamster.
Biol. Reprod.
 
8
,
378
–387.
Lamperti, A. A., and Printz, R. H. (
1974
). Localization, accumulation, and toxic effects of mercuric chloride on the reproductive axis of the female hamster.
Biol. Reprod.
 
11
,
180
–186.
Lamperti, A. A., and Niewenhuis, R. (
1976
). The effects of mercury on the structure and function of the hypothalamo-pituitary axis in the hamster.
Cell. Tissue Res.
 
170
,
315
–324.
Magos, L. (
1967
). Mercury blood interaction and mercury uptake by brain.
Environ. Res.
 
1
,
323
–337.
Marinova, G., Chakarova, O., and Kaneva, Y. A. (
1973
). A study of reproductive function of women working with mercury.
Probl. Akush. Ginekol.
 
1
,
75
–77.
Mikhailova, L. M., Kobyets, G. P., Lyubomudrov, V. E., and Braga, G. F. (
1971
). The influence of occupational factors on diseases of the female reproductive organs.
Pediatr. Akush. Ginekol.
 
33
,
56
–58.
Panova, Z., and Dimitrov, G. (
1974
). The ovarian function in women with occupational exposure to metallic mercury.
Akush. Ginekol.
 
13
,
29
–34.
Rothstein, A., and Hayes. A. D. (
1960
). The metabolism of inhaled mercury in the rat studied by isotope techniques.
J. Pharmacol. Exp. Ther.
 
138
,
1
–10.
Rowland, A. S. (
1994
). The effect of occupational exposure to mercury vapor on the fertility of female dental assistants.
Occup. Environ. Med.
 
51
,
28
–34.
Schuurs, A. H. B. (
1999
). Reproductive toxicity of occupational mercury. A review of the literature.
J. Dentistry
 
27
,
249
–256.
Sikorski, R., Jiszkiewicz, T., Paszkowski, T., and Szprengier-Juszkiewicz, T. (
1987
). Women in dental surgeries: Reproductive hazards in occupational exposure to metallic mercury.
Int. Arch. Occup. Environ. Health
 
59
,
551
–557.
Sokal, R. R., and Rohlf, F. J. (1969). Biometry. The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Co., San Francisco.
Stockwell, P. B., and Corns, W. T. (
1993
). The role of atomic fluorescence spectrometry in the automatic environmental monitoring of trace element analysis.
J. Automatic Chem.
 
15
,
79
–84.