Maternal long-term inhalation exposure to perchloroethylene and prenatal teratogenicity: morphometric, hormonal, and histological study

Abstract Some commonly used chemicals have teratogenic effects. Perchloroethylene (PCE) is a liquid that is widely used in various industries and drying clothes. In this study, the teratogenic effects of PCE in rat embryos were investigated. In this experimental study, 32 adult Wistar female rats in the weight range of 230-250 g were used. Female rats were randomly divided into 4 groups (n = 8). Control group (without PCE inhalation), experimental group G(I) (exposed to PCE 18 days prior to mating), experimental group G(II) (exposed to PCE 18 days after mating) and experimental group G(III) (exposed to PCE 18 days before and 18 days after mating). Pregnant rats were anesthetized on the 18th day of gestation and then serum and embryos were removed for the required studies. Embryos were examined for number, weight, sex, morphometric parameters of organs, and tissue samples were prepared for histological studies. Serum isolated from dams were evaluated for sexual and gonadal hormones. The results of this study showed that PCE has teratogenic effects on rat embryos. Infertility and reduced birth rate were other effects of PCE in rats. PCE has teratogenic effects and impairs the reproductive system of rats.


Introduction
Numerous factors can interfere in the development of the fetus and can cause malformations. Such factors are called teratogens. Teratogenic agents have a wide range as such that some of them exist in nature and others are created by different industries (Flamier et al. 2017). Teratogens, in addition to causing morphometric abnormalities in fetal organs, can in some cases cause various types of cancer, whether benign or malignant, in various tissues of the body (Xing et al. 2017). There have been numerous reports of infertility, fertility with a deformed fetus, and abortion due to teratogenic effects (Rasmussen 2012). In the United States, 4% of infants are born with congenital anomalies, of which 20% usually result in death annually (Worley et al. 2018). Many teratogenic agents are of chemical origin and are produced by industry. Excessive use of such substances or using them without adequate information or knowledge have created many problems in human society and public health (Liu et al. 2020). One of the most widely used chemicals in the cleaning industry is perchloroethylene (tetrachloroethylene). Perchloroethylene (PCE) is a widely used chlorine solvent that is widely adopted in the laundry industry, textile processing, and metal cleaning operations (Vlaanderen et al. 2014). PCE is a colorless, ether-like liquid with a sweet taste. PCE was used in the early twentieth century to treat hookworm infections (Cichocki et al. 2016). It has been reported that this substance can cause various abnormalities through drinking water, inhalation of its gas from the air, and even its consumption in food pollution (Habib et al. 2018). PCE interferes with human reproduction and causes infertility and impaired production of active sperm in men (Perrin et al. 2007). One study found that infants or children exposed to PCE experienced visual impairment in adulthood (Getz et al. 2012). In a study of a rare disease or male breast cancer, it was found that men exposed to solvents such as PCE or heavy metals were 25-20% more likely to develop the disease than other men (Jones 2011). Today, the need of people for laundry workshops is increasing. Dry cleaning is a way to clean fabrics without the use of water. This method is used to wash the fabric without damaging its fibers. The most popular chemical used by dry cleaners is PCE. In centers which use PCE, its gas is widely released into the air, exposing people, especially laundry workers, to toxicity (McKernan et al. 2008). A report has shown that in some residential complexes that use PCE, even its volatile gas can be effective in creating intermediate contamination among the residents (McDermott et al. 2005). Since the harmful effects, especially the teratogenic effects of PCE on the fetus, have not been previously probed, using morphometric, hormonal, and histological analyses, the present research is aimed at investigating such effects for the first time in rats as well as fetuses exposed to PCE.

Animals
In this experimental study, 32 adult female Wistar rats weighing 230-250 g were purchased from Hamadan University of Medical Sciences, Iran. Female virgin rats were mated overnight with male rats (one female for one male). The day evidence for positive mating was manifested was recorded as gestation day (GD) 0. The mating females were stored separately in rat cages of stainless steel. During the study, food and potable water were freely available to the animals, except at the time of inhalation. The ethics of working with animals were observed throughout the whole study in accordance with the international standards. The on-site ethics committee also approved the research (approval number: IR.BASU.REC.1398.021).

Experimental design
PCE (CAS Registry Number: 127-18-4) substance was obtained from Inovyn company (France) with purity >99.9% according to the seller's receipt. All animals were exposed to 100 ppm PCE for 6 h a day (Carney et al. 2006). The rats were randomly divided into 4 groups (via simple randomization): Control group: In this group, there were 8 adult female rats with free access to water and food. Experimental group 1 (G(I)): In this group, 8 female rats were placed in a special chamber and exposed to PCE at a concentration of 100 ppm for 18 days. Consequently, the rats were mated by a male. Experimental group 2 (G(II)): In this group, 8 adult female rats were placed with a male rat for mating similar to experimental group 1. When mating (vaginal plaque examination) and pregnancy were confirmed, they were exposed to PCE at a concentration of 100 ppm for 18 days. Experimental group 3 (G(III)): In this group, 8 female rats were exposed to PCE at a concentration of 100 ppm, 18 days before pregnancy and 18 days after it.
The concentration dose of 100 ppm was based on previous reports in animal and human exposed to PCE (Carney et al. 2006; WHO Guidelines Approved by the Guidelines Review Committee 2010). After testing on the 18th day of pregnancy, all rats were anesthetized with ketamine hydrochloride (50 mg/kg body weight) and xylazine (10 mg/kg body weight); then, tissue and embryos' samples were extracted. Also, blood samples taken from the inferior vena cava of female rats were collected. The embryos were first weighed and morphometric characteristics such as body dimensions, head, body length, as well as arm and leg length were examined. The embryos were then placed in 10% formalin for histological examination at a rate of 10 times that of the sample size for fixation.

Hormonal measurement
After one hour, the blood samples of female rats were centrifuged for 10 min at 3,000 rpm and their serum was isolated to measure the levels of hormones. ELISA Kit (ZellBio GmbH, Germany) and ELISA reader (BioteTek ELx808, USA) were used to measure serum levels of LH, FSH, progesterone (P 4 ) and estradiol (E 2 ). The sensitivity of the kit to LH, FSH, testosterone, and E 2 was 0.05 mIU/ml, 0.12 mIU/ml, 0.02 ng/ml, and 4.45 pg/ml, respectively. Moreover, the coefficients of variation (CV) of these hormones were less than 10 according to the manufacturer's instructions.

Histologic studies
Fetal skin (provided from the dorsal area in all fetuses), lung, liver, and kidney tissue slides were prepared for a more indepth examination of tissues and microscopic studies. After one week and stabilization of the samples, the samples were placed in separate baskets and encoded in the tissue processing machine to perform the tissue processing steps. These steps were performed automatically by this device. After tissue stabilization, the paraffin-embedded tissues were ready for molding. Tissue blocks were prepared by a microtome machine with 5 mm slices of tissue. Then, in order to open the wrinkles of the tissues, it was placed in a floating tissue machine with a temperature of 40 C; the tissues were then transferred to stained glass slides for staining. Stained tissue slides were examined under an optical microscope. Then, photographs were taken from tissue sections using a digital microscope.

Special exposure chamber
In order for the tested rats to be exposed to air with a certain concentration of PCE, a chamber made of stainless steel and glass was prepared. In this chamber, the cages of the tested rats, except for the control group, were placed. During the experiments, the animals were exposed to dynamic airflow in 0.75 cubic meters of stainless steel and glass, and the rats were placed in this chamber for 6 h to face the substance in the air and breathe from it. The chamber air flow was retained at approximately 150 L/min, which was sufficient to maintain the normal oxygen concentration. PCE concentrations were measured several times daily using the GC analysis. After the experiment, the rats were transferred to the animal room under normal conditions (Carney et al. 2006).

Statistical method
Data were analyzed using GraphPad Prism software version 9. Kolmogorov-Smironov test was used for testing the normality of data. Then one-way analysis of variance was run to examine within the subject comparisons. For the comparisons between groups, Tukey post hoc test was used for parametric data and Kruskal-Wallis test was used for non-parametric data. The level of significance was considered at p < 0.05.

Morphometric characteristics of maternal, and fetal
from different experimental groups exposed to PCE Morphometrical changes of maternal and fetal from different groups affected by PCE are shown in Table 1 (see also Supplementary Figure 1(a-d)). The comparison of morphometric characteristics in relation to body length, arm length, leg length, head length, body weight, and number of embryos demonstrated that the teratogenic effects of PCE in the development of embryonic organs and the number of embryos formed were significantly different compared with the control group. The differences amongst the examined groups with each other also indicated the fact that experimental group G(III) had the most damage to its fetuses. The results showed that there was no significant difference between the control group and experimental group G(I) except for fetal leg and head length and fetal body width. However, a significant decrease in experimental group G(II) and experimental group G(III) was observed compared to the control group. Moreover, the change in the body length of embryos in experimental group G(II) to experimental group G(I) was significantly reduced. In addition, the difference between experimental group G(III) with experimental groups G(I) and G(II) was significantly diminished (Table 1).
3.2. Histological examination of fetal from different experimental groups exposed to PCE Histopathological examinations of embryos from pregnant rats affected by PCE are shown in Figures 1-4. Skin tissue from the normal control group shows it is normally developed. The epidermis contains all typical layers from the stratum basale toward the cornified layer of stratum corneum. The underneath layer of dermis has normal structure with growing hair follicles (Figure 1(a)). Skin tissue from G(I) and G(II) groups shows the skin layers of the epidermis and dermis are slightly thinner than normal, along with fewer number of developing hair follicles (Figure 1(b,c)). Skin tissues from G(III) group showed that the epidermal and dermal layers of the skin are thinner than normal with underdeveloped hair follicles (Figure 1(d)). Lung tissues from the normal control group showed that lung tissue has normal histology with developing bronchial ducts and alveoli (Figure 2(a)). Lung tissues from G(I) and G(II) groups showed that the microscopic architecture of the lung is normal with developing airways and alveoli ( Figure  2(b,c)). Furthermore, lung tissues from G(III) group showed that the lung has fewer bronchial ducts and alveoli in a condensed interstitium, probably in association with delayed growth of the embryos (Figure 2(d)).
Liver tissues from the normal control group showed that the microscopic structure of the liver is normal with central venules in the middle of the developing lobules along with many sinusoidal capillaries (Figure 3(a)). Liver tissues from G(I) and G(II) groups showed that the liver has normal parenchyma, however, with relatively slow development of lobules (Figure 3(b,c)). Liver tissues from G(III) group showed that the liver is small and structurally undeveloped, with no detectable lobules and vessels (Figure 3(d)).
Kidney tissues from the normal control group showed that the kidney has normal histology with renal corpuscles mainly in the cortex, and developing renal tubules inside the organ interstitium (Figure 4(a)). Kidney tissues from G(I) and G(II) groups showed that the kidney is normally developed with renal corpuscles and renal tubules (Figure 4(b,c)). Additionally, kidney tissues from G(III) group showed that the kidney is smaller than normal with smaller renal corpuscles and underdeveloped medulla (Figure 4(d)).
3.3. Hormonal evaluation of maternal rats from different experimental groups exposed to PCE From the results obtained from measuring the hormonal plasma level in the maternal experimental groups, it was found that there was a significant decrease in LH, FSH, P 4 , and E 2 levels between the experimental groups compared with the control group. Moreover, while there was also a Values represent mean ± standard deviation. significant decrease between experimental groups G(II) and G(III) compared to experimental group G(I), in the E 2 level, there was no significant difference between experimental groups in terms of LH/FSH ratio except in P 4 /E 2 ratio between G(II) and G(III) (Figures 5(a-f)).

Discussion
The results of this study showed that PCE as a chemical substance can act as a teratogenic agent and cause morphometric abnormalities in the embryos of adult female rats and even cause various diseases in such adult rats. Adult female rats exposed to inhalation of PCE at different times-i.e., before pregnancy or during pregnancy or before and after pregnancyin addition to morphometric and histological abnormalities in various tissues, experienced weight changes. Morphometric changes include reduction in fetal arm and leg size, reduction in fetal head length and width, fetal weight loss as well as weight loss in adult female rats, fetal tail length reduction, and reduction in the size of the body from the snout to the end of the tail in the fetus.
During pregnancy, the embryo and fetus in the uterus are very sensitive to the parameters of their surrounding environment. It can even be said that the body of the pregnant mother is more sensitive than before pregnancy. Some teratogens cross the fetal placental barrier and cause adverse effects on the developmental stages of the embryo or fetus (Rasmussen 2012;Xing et al. 2017). In another study in Yazd, Iran, fifty-nine workers who were exposed to PCE for at least three months in laundry rooms, were examined. Examination of these workers showed relatively large initial DNA damage in these workers (Azimi et al. 2017). During the oxidation of PCE, cytochrome P450 produces trichloroethanol, trichloroacetyl chloride, and trichloroacetic acid (TCA). A metabolite of PCE, TCA has hepatocarcinogenic effects on rodents as well as humans. In addition to lipid peroxidation, TCA causes oxidative DNA damage; these processes have been implicated in the development of cancer (Toraason et al. 2003). Considering these results, it can be concluded that perhaps one of the reasons for changes in morphometric and morphogenetic parameters in embryos is due to changes in their DNA. In another study that was performed on rats and rabbits with different time ranges of 1-19 days and 1-24 days, Figure 1. Histopathological examination of the skin tissue of embryos from pregnant rats affected by PCE. Skin tissue from normal control group. Skin is normally developed. The epidermis contains all typical layers from the stratum basale (large arrow) toward the cornified layer of stratum corneum (small arrow). The underneath layer of dermis (asterisk) has normal structure with growing hair follicles (arrowhead) (a). Skin tissue from G(I) group. Skin layers of the epidermis (large arrow) and dermis (asterisk) are slightly thinner than normal, along with fewer number of developing hair follicles (small arrow) (b). Skin tissue from G(II) group. Skin layers of the epidermis (large arrow) and dermis (asterisk) are slightly thinner than normal, along with fewer number of developing hair follicles (small arrow) (c). Skin tissue from G(III) group. Epidermal (arrow) and dermal (asterisk) layers of skin are thinner than normal with underdeveloped hair follicles (arrowheads) (d).
respectively, tetrachloroethylene was observed to cause teratogenic effects in these animals; these teratogenic effects were seen more in the embryonic period of these animals (Hardin et al. 1981). Another study looked at 2,999 women who were directly exposed to PCE during pregnancy and the fetuses of 1,658 children who were exposed to PCE during pregnancy. The results of this study showed that children who were exposed to PCE during their mother's pregnancy showed more abnormalities compared to other children who were not exposed to this substance during their mother's pregnancy (Aschengrau et al. 2009).
Another important result of this study is that fetuses exposed to PCE had different abnormalities in different tissues including skin, lung, liver, and kidney compared to fetuses in the control group. This is most likely due to the lack of healthy development and growth or because of insufficient development and unsuccessful organogenesis. This particular finding can be inferred from another study as well. In this study, due to continuous and intermittent inhalation of PCE, the weight of the limbs and their morphological shape were subject to alteration. Exposure of adult female rats for 30 days resulted in weight loss in the embryos as well as weight loss in the kidneys and liver at high concentrations of PCE (NTP Toxicology and Carcinogenesis Studies of Tetrachloroethylene (Perchloroethylene)  in F344/N Rats and B6C3F1 Mice (Inhalation Studies) 1986).
Another important result of this study is that in different groups, the number of embryos in adult female rats has a reverse relationship with the duration of their exposure to PCE. That is, in the group that was exposed to PCE for a longer period of time, there was no fertility in some cases, and in the groups that were exposed to PCE for a shorter period of time, the number of embryos was lower than in the control group. Other studies have suggested that PCE may cause abortion or reduced fertility due to macroscopic abnormalities in the developmental mechanisms. In order to investigate the relationship between spontaneous abortion and work in laundry units in the United Kingdom where PCE solvent was used, the rate of spontaneous abortion and reproductive abnormalities was 78% for workers who were currently working, but in workers who were former workers was 46% (Doyle et al. 1997). In another study that was consistent with this study, the rate of fertility and spontaneous abortion in laundry workers who were exposed to PCE was investigated. Exposure to PCE also increased the rate of Figure 2. Histopathological examination of the lung tissue of embryos from pregnant rats affected by PCE. Lung tissue from normal control group. Lung tissue has normal histology with developing bronchial ducts (large arrows) and alveoli (small arrows) (a). Lung tissue from G(I) group. The microscopic architecture of the lung is normal with developing airways (large arrows) and alveoli (small arrows) (b). Lung tissue from G(II) group. The microscopic architecture of the lung is normal with developing airways (large arrow) and alveoli (small arrow) (c). Lung tissue from G(III) group. Lung has fewer bronchial ducts (large arrows) and alveoli (small arrows) with the condensed interstitium (asterisk), probably in association with delayed growth of the embryos (d). Hematoxylin & Eosin, Â100 magnification, Scale bar ¼ 100 mm.
spontaneous abortions compared to the same group (Eskenazi et al. 1991). In a study on chicken embryos, chlorinated compounds such as PCE were used. On the 14th day of incubation, PCE gas was injected into the environment where the fertilized eggs were located; then, different parameters of the chicken embryo were examined. In this study, mortality was significantly increased; moreover, macroscopic abnormalities were also observed (Elovaara et al. 1979). These results indicate that PCE has adverse effects on the growth and development of various organisms.
Other findings of the present study include significant changes in sex hormones in adult female rats. PCE, due to its effect on dopamine depletion, can be attributed to a significant decrease in sex hormones, menstrual irregularities, and a significant decrease in gonadotropins due to disorders caused by PCE in the hypothalamus-pituitary-gonadal axis (Beliles 2002). Another negative effect of PCE, which is a decrease in sex hormones (that may have even led to infertility), is the imitation of estrogen and androgen hormones. In a study conducted to investigate the effects of occupational and environmental factors on endocrine function, important results were found raising concerns. This was due to the fact that in addition to mimicking estrogen and androgen hormones and disrupting hormonal pathways, they were also observed to mimic other hormones and cause disorders in other glands (Alofe et al. 2019). Significant reductions in these hormones owing to the exposure to inhalation in rats in the present study can have many adverse effects including spontaneous abortion, infertility as one of the most likely ramifications. Our study has some limitations that should be considered. As limitations, our study did not carry out an experimental part to evaluate the development of the offspring due to limited funding, and time constraint. Therefore, further studies are required to determine whether possible changes observed during embryonic development can have similar impacts on postnatal development and adulthood.

Conclusion
The results of the present study showed that PCE is a teratogenic substance and has adverse effects on the development of the embryo or fetus and causes congenital anomalies after childbirth. Placement of adult female rats during pregnancy with the daily exposure of 100 ppm of PCE causes teratogenic effects in the fetuses of adult female rats. Such Figure 3. Histopathological examination of the liver tissue of embryos from pregnant rats affected by PCE. Liver tissue from normal control group. The microscopic structure of the liver is normal with central venules (large arrows) in the middle of the developing lobules along with many sinusoidal capillaries (small arrows) (a). Liver tissue from G(I) group. Liver shows normal parenchyma, however, with relatively slow development of lobules (b). Liver tissue from G(II) group. Liver shows normal parenchyma, however, with relatively slow development of lobules (c). Liver tissue from G(III) group. Liver is small and structurally undeveloped (asterisk), with no detectable lobules and vessels (d). Hematoxylin & Eosin, Â100 magnification, Scale bar ¼ 100 mm. . Histopathological examination of the kidney tissue of embryos from pregnant rats affected by PCE. Kidney tissue from normal control group. Kidney shows normal histology with renal corpuscles (large arrow) mainly in the cortex, and developing renal tubules (arrowhead) inside the organ interstitium (a). Kidney tissue from G(I) group. Kidney is normally developed with renal corpuscles (large arrow) and renal tubules (small arrow) (b). Kidney tissue from G(II) group. Kidney is normally developed with renal corpuscles (large arro)) and renal tubules (small ar)ow) (c). Kidney tissue from G(III) group. Kidney is smaller than normal with smaller renal corpuscles (arrow) and underdeveloped medulla (asterisk) (d). Hematoxylin & Eosin, Â100 magnification, Scale bar ¼ 100 mm. Figure 5. Comparison of the serum LH (a), FSH (b), LH/FSH ratio (c), P 4 (d), E 2 I and P 4 /E 2 ratio (f) levels in the control and PCE affected groups. Data are shown as median (min, max) of eight animals per group. Ã p < 0.05, ÃÃ p < 0.01, and ÃÃÃ p < 0.001vs. control group. ### p < 0.001 vs. G(I) group. † † † p < 0.001 vs. G(II) group. ramifications include skin, lung, liver, and kidney deformities and most importantly reduction of the number of embryos in adult female rats. This also can even indicate the lack of fertility in adult female rats. Other effects of this substance include a significant reduction in the levels of sex hormones and gonadotropins in the serum of adult female rats. exposure and bladder cancer risk: a meta-analysis of dry-cleaningworker studies. Environ Health Perspect. 122 (7)