Combined Female Reproductive Toxicity Of Representative Chemicals Involving Phthalates Acid Esters And Polycyclic Aromatic Hydrocarbons And Health-based Risk Assessment

Background and objectiveNowadays, expanding urbanization and industrialization has led to ubiquitous chemical contaminants in environment that have clearly been defied as endocrine disrupting chemicals and these problems have been received much more concern by scientists. Persistent organic pollutants (POPs) are chemical substances that persist in the environment, bioaccumulate through the food web, and subsequently pose potential risks to human health. Many research studies showed that POPs pollution is becoming the most serious problem all over the world. The main pollution chemicals of POPs in the water body of the Three Georges reservoir was characterized by phthalates acid esters (PAEs) and polycyclic aromatic hydrocarbons (PAHs). Phthalates acid esters and polycyclic aromatic hydrocarbons have clearly been defied as endocrine disrupting chemicals, a great deal of literatures indicates that these environmental toxicants may accumulate in ovary and cause reproductive disorders. Traditionally, reproductive studies have focused on healthy effects associated with exposure to single environmental endocrine disrupting chemical, but exploration of interactions among these chemicals is an important and also key area of enquiry. The toxicity of chemical mixtures might be lower or higher than predicted from the known individual effects of each chemical, which could be a synergistic, antagonistic, potentiation or inhibitory interaction in any mixture-related studies. In addition, adult women, children, particularly neonates, can be biologically more sensitive to the mixture toxicants exposure on physiological functions and characteristics basis.In our studies conducted in the Three Gorges reservoir, the higher average concentrations of PAEs and PAHs chemicals were found in aquatic environment, thus dietary exposure to these chemicals is possible which can occur simultaneously. Taking these facts into consideration, the present study was divided into three parts. In terms of part one and two, we chose the representative chemicals of PAEs and PAHs, namely DEHP and B[a]P separately, also their active metabolites MEHP and BPDE, as the model chemicals to evaluate the combined female reproductive toxicity as well as their possible mechanism in vivo and in vitro study. The third part introduced the health risk assessment method and established health risk assessment model for POPs contamination in water environmental so as to explore potential healthy hazards of POPs in the Three Gorges reservoir. The purpose of this study is to evaluate the combined reproductive toxicity and involved mechanism, provide experiment data and explore a method to evaluate the adverse effect of organic pollutants mixture on human health.Materials and MethodsPartⅠFemale reproductive toxicity following DEHP and B[a]P combination.1. This study was to determine the female reproductive toxicity and possible molecular mechanism of oral exposure to DEHP and B[a]P combination following 30 and 60days using Sprague-Dawley (SD) rat's model. Females SD rats were randomly assigned to seven groups of 12 animals at each group. Female rats were given intragastrical administration of corn oil (control), B[a]P alone (B[a]P5mg/kg and B[a]P10mg/kg), DEHP alone (DEHP300mg/kg and DEHP600mg/kg) and B[a]P+DEHP (B[a]P5mg/kg+DEHP300mg/kg and B[a]P10mg/kg+DEHP600mg/kg) on alternate days for both 30 and 60days.2. After the toxical exposure, rats were killed and general clinical observation, reproductive organs, serum hormone level, ovarian follicle population, cell apoptosis, ovary histopathology, mRNA levels of target genes and proteins expression including PPARs, P450Arom and 17β-HSD in granulosa cells were evaluated.PartⅡCytotoxicity of MEHP+BPDE in cultured granulosa cells1. Obtain and identify the cultured granulosa cells from the ovary of impuberism rats so as to establish a convenient and stable cell experiment model.2. The doses of MEHP and BPDE intoxication was chosen and 3×3 factorial design model in vivo study was established, As detailed as below, Control (DMSO), MEHP(25μM; 50μM), BPDE(10nM; 100nM), MEHP+BPDE (25μM+10nM; 25μM+100nM; 50μM+10nM; 50μM+100nM). Cytotoxicity of MEHP or BPDE alone and concomitant treatments was evaluated by MTT and histopathology observation.3. The target genes and proteins expression including P450Arom and PPARs in cultured granulosa cells were evaluated using RT-PCR and immunohistochemistry methods. PartⅢWater environmental health risk assessment of PAEs and PAHs.1. Seasonal samplings were carried out in Feb and Aug of 2005. Water samples were collected from seven different sampling sites of trunks and tributaries in the Three Gorges reservoir. The organic pollutants samples were analyzed by GC/MS.2. U.S.EPA'S model recommended for water environmental health risk assessment was improved based on the water monitoring data in the year of 2005. The health risk assessment method was introduced for organic pollutants contamination in water environment and selected 6 kinds of organic pollutants (PAEs and PAHs), which is DBP, DEP, DEHP, NA, Pyr and FLA, to carry out data analysis and evaluate the total health risk.ResultsPartⅠIn vivo study1. The growth and development of rats in DEHP and B[a]P alone and combination groups was not were obviously inhibited compared with control (P>0.05). The absolute ovary weight and ovary/body weight ratio of rats exposed to B[a]P+DEHP treatment for 30 and 60 days was significantly decreased compared to control (P<0.05), dose- and time-response tendency appeared. The joint effect of B[a]P+DEHP on ovary/body weight ratio appeared no interaction.2. DEHP and B[a]P alone or combination resulted in prolonged estrous cycle as well as shorten estrus phase in animals following 30 and 60 days exposure compared to control (P<0.05). After 60 days exposure, B[a]P and DEHP alone or combination (besides B[a]P5mg/kg) resulted in prolonged EC and No E (P<0.01), the estrous phase of B[a]P10mg/kg+DEHP600mg/kg and DEHP600mg/kg treated groups was shortened (P<0.05), there was significant difference between B[a]P10mg/kg+DEHP600mg/kg and B[a]P10mg/kg or B[a]P5mg/kg+DEHP300mg/kg (P<0.01). The joint effect of B[a]P+DEHP on EC and E appeared no interaction.3. It showed that B[a]P+DEHP caused remarkable changes in serum hormone (E2 and P) levels following 30 day exposure. B[a]P (besides B[a]P5mg/kg) or DEHP alone and B[a]P+DEHP significantly reduced the serum E2 ,P and T levels following 60 days exposure compared to the control (P<0.05), serum E2 level was significantly decreased in B[a]P+DEHP compared to B[a]P intoxication (P<0.05). It seemed there was no significant changes in terms of serum FSH and LH levels in either following 30 or 60 days exposure (P>0.05). The joint effect of B[a]P+DEHP on serum E2 and P appeared no interaction.4. B[a]P+DEHP resulted in decreased primordial and primary/secondary follicle populations, and increased atretic populations. After 30 days exposure, B[a]P or DEHP alone (besides DEHP300mg/kg) and B[a]P+DEHP had significantly reduced primordial follicle populations of ovaries compared to control (P<0.05). After 60 days exposure, it was shown that only the primordial populations significantly decreased in B[a]P+DEHP and B[a]P10mg/kg compared to control (P<0.05), The primordial follicle populations of B[a]P+DEHP significantly decreased compared to DEHP alone (P<0.05). The atretic numbers in the high dose of DEHP and B[a]P+DEHP significantly increased (P<0.05). The joint effect of B[a]P+DEHP on primordial follicles, primary/secondary follicles, atretic follicles and corpora lutea appeared no interaction.5. B[a]P and DEHP alone or combination resulted in granulosa cell structure damage. cellular structure rarefaction, exfoliation and degeneration was observed in any treated groups under light microscope, additionally, the interspaces between granulosa cells and theca cells widened in B[a]P and B[a]P+DEHP treated groups. B[a]P+DEHP caused wide spread cellular swelling and lysis of plasma membranes. Mitochondrial cristae appeared swelling and collapse, cellular myelin figure and apoptotic body was observed with electron microscope. Accompanied by exposure extension, cellular damage was more serious, especially focal necrosis and myelin figure was observed frequently.6. Whenever following 30 or 60 days exposure, B[a]P or DEHP alone (except DEHP300mg/kg) and B[a]P+DEHP dramatically increased the granulosa cells apoptosis in ovary compared to control (P<0.05), caspase-3 activation involved in the process of apoptosis occurrence (P<0.05). Remarkable increase of granulosa cell apoptosis was found in B[a]P+DEHP, however, cell apoptosis index decreased while following 60 days exposure. There was high correlation between apoptosis and atretic follicle occurrence. The joint effect of B[a]P+DEHP on granulosa cells apoptosis and caspase-3 expression appeared obvious interaction (P<0.05).7. The target gene expression of P450Arom and PPARs was in coincidence with immunohistochemical and western blot results in granulosa cells. The P450Arom mRNA level and protein expression was obviously down-regulated in DEHP or B[a]P alone and combination groups compared to control (P<0.05), DEHP and B[a]P+DEHP treatment significantly up-regulated 17β-HSDΠmRNA level (P<0.05). In the two phase points of dose-dependent study, pronounced increases in PPAR-αand PPAR-γversus control were observed in any treatment groups (P<0.05). It was shown that the down-regulation of P450Arom was highly correlated with up-regulation of PPARs, DEHP played an important role in the combined mechanism of ovotoxic action. The joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05).PartⅡIn vitro study1. More than 95%of highly purified granulosa cells could be obtained by mechanical method combined with trypsin digestion and brachytely centrifugation, the exponential phase of growth was between 48h and 96h incubation. Moreover, identifications of granulosa cells by HE and FSHR staining were volant and convenient approaches.2. It showed that both MEHP and BPDE induced cytotoxicity in cultured granulosa cells, MEHP 200μM incubation for 24h obviously inhibited granulosa cells proliferation, and the viability of the cells were significantly inhibited after BPDE 10nM incubation for 24h (P<0.05).3. MEHP+BPDE incubation for 12h and 24h, to a certain extent, inhibited granulosa cell viability compared to control (P<0.05), the joint effect of B[a]P+DEHP appeared no interaction, which suggesting addition joint action of the two compounds. Additionally, BPDE and MEHP+BPDE incubation for 24h resulted in significant cell damage as revealed by nuclear atrophy and cellular vacuolation and cellular structural damage, such as cell layer withdrawal and destruction.4. MEHP or BPDE alone and combination, to a certain extent resulted in down regulation of P450Arom mRNA and protein expression, MEHP+BPDE significantly up regulated the PPAR-γmRNA and protein expression compared to control (P<0.05), the joint effect of B[a]P+DEHP on P450Arom and PPAR-γprotein expression appeared obvious interaction (P<0.05), which suggesting a synergistic action of the two compounds.PartⅢWater environmental health risk assessment1. Organic compounds were detected in water samples from the Three Gorges reservoir,which included 178 kinds of POPs in winter and 144 in summer. 18 kinds of POPs can be found on the EAP's organic pollutants blacklist, 7 kinds of POPs can be found on the blacklist of Chinese government. The concentration of 6 kinds of pollutants (DBP, DEP, DEHP, NA, Pyr, FLA) in this study varied from ND～3.60×10-3.2. The results showed that the greatest health risk to the individual person per year in terms of 6 kinds of organic pollutants through drinking is DEHP, followed by DBP, Pyr, NA, FLA and DEP, the health risk of the above organic pollutants varied from 6.34×10-14 a-1 to3.99×10-11 a-1. The average total health risk caused by main organic pollutants (PAEs and PAHs) was 8.86×10-12 a-1 and even lower than the maximum tolerable value (5.0×10-5·a-1) recommended by ICRP. The most heavily polluted section located in Cuntan area among all the sampling sections and might have higher health risk.Conclusion1. DEHP and B[a]P, also their active metabolites MEHP and BPDE, possessed female reproductive toxicity, granulosa cell was the principal and common target cell of the cytotoxicity.2. DEHP and B[a]P combination obviously exerted ovotoxicty in rats through decreasing ovary/body weight ratio, causing prolonged estrous cycle and shorten estrus, suppressing sex hormone levels, decreasing ovarian follicle populations, increasing atretic follicles and damaging the cell structure. The joint effect of DEHP and B[a]P combination on the above parameters appeared no interaction, DEHP played the principal role in the toxic actions. MEHP and BPDE combination inhibited granulosa cell viability and resulted in significant cell damage as revealed by nuclear atrophy, cellular vacuolation and cellular structural damage. The joint effect of DEHP and B[a]P combination appeared no interaction, which suggesting addition joint action of the two compounds.3. DEHP and B[a]P combination obviously induced granulosa cells apoptosis, caspase-3 activation involved in the process of apoptosis occurrence, it was shown that atretic follicles were closely correlated with cell apoptosis. The joint effect of DEHP and B[a]P combination on granulosa cells apoptosis and caspase-3 expression appeared interaction.4. According to in vivo and in vitro study, it is demonstrated that B[a]P+DEHP and MEHP+BPDE activated PPARs signaling pathway and then down-regulated P450Arom expression, subsequently, the balance of sex hormone synthesis, secretion and homeostasis was destroyed. As for interactive effect of the combination mechanism, we draw a conclusion that synergistic effects were observed in P450Arom and PPARs protein expression.5. The results showed that PAEs and PAHs pollution was widespread in aquatic environment of the Three Gorges reservoir. The total health risk caused by the six kinds of organic pollutants (PAEs and PAHs) was in low level. However, it also should be paid more attention by our environment protection management.