Pentachlorophenol inhibits CatSper function to compromise progesterone's action on human sperm.

Pentachlorophenol (PCP), a highly toxic contaminant of chlorophenols, is common in a variety of environments and presents serious risks to animal and human health. However, the reproductive toxicity and potential actions of PCP have not been investigated thoroughly, especially in humans. Here, human spermatozoa were used to evaluate the effect of PCP on cell function and to explore the underlying mechanisms. PCP had no substantive effects on sperm viability or motility, nor on the ability to penetrate viscous medium, sperm hyperactivation or spontaneous acrosome reactions. However, PCP significantly inhibited these properties induced by progesterone (P4). Consistent with the functional observations, although PCP itself did not affect the basal intracellular Ca2+ concentrations and CatSper current, PCP dose-dependently inhibited increases of intracellular Ca2+ concentrations caused by P4. In addition, the activation of CatSper induced by P4 was largely suppressed by PCP. This is the first report showing that PCP may serves as an antagonist of the P4 membrane receptor to interfere with Ca2+ signaling by compromising the action of P4 on regulating sperm function. These findings suggest that the reproductive toxicity of PCP should also be a matter of concern as a mammalian health risk.

Metabolic Activation and Cytotoxicity of Aloe-Emodin Mediated by Sulfotransferases.

Aloe-emodin (AE) is a major anthraquinone ingredient of numerous traditional Chinese medicines with a variety of beneficial biological activities in vitro. Previous studies suggested that AE possessed cytotoxicity and genotoxicity. Nevertheless, the mechanisms of the toxic action of AE have not yet been fully clarified. The present study aimed at characterization of metabolic pathways of AE to better understand the mechanisms of AE-induced cytotoxicity. An AE-derived glutathione conjugate (AE-GSH) was observed in rat liver cytosol incubations containing AE and GSH, along with 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Similar incubation fortified with N-acetylcysteine (NAC) in place of GSH offered an AE-NAC conjugate corresponding to the GSH conjugate. The formation of the two conjugates was found to require PAPS. The two conjugates were respectively detected in bile and urine of rats given AE. Sulfotransferase (SULT) inhibitor pentachlorophenol (PCP) suppressed the production of the observed AE-GSH/NAC conjugates in vivo, which suggested that SULTs participated in the process of the metabolic activation of AE. The presence of PCP attenuated cell susceptibility to AE-induced cytotoxicity. The present study illustrated potential association of sulfation-mediated bioactivation of AE with its cytotoxicity.

Effects of inhibition of hepatic sulfotransferase activity on renal genotoxicity induced by lucidin-3-O-primeveroside.

Sulfotransferase 1A (SULT1A) expression is lower in the liver of humans than that of rodents. Therefore, species differences should be taken into consideration when assessing the risk of rodent hepatocarcinogens metabolically activated by SULT1A in humans. Although some renal carcinogens require SULT1A-mediated activation, it is unclear how SULT1A activity in the liver affects renal carcinogens. To explore the effects of SULT1A activity in the liver on genotoxicity induced by SULT1A-activated renal carcinogens, B6C3F1 mice or gpt delta mice of the same strain background were given lucidin-3-O-primeveroside (LuP), a hepatic and renal carcinogen of rodents, for 4 or 13 weeks, respectively, and pentachlorophenol (PCP) as a liver-specific SULT inhibitor, was given from 1 week before LuP treatment to the end of the experiment. A 4 week exposure of LuP induced lucidin-specific DNA adduct formation. The suppression of Sult1a expression was observed only in the liver but not in the kidneys of PCP-treated mice, but co-administration of PCP suppressed LuP-induced DNA adduct formation in both organs. Thirteen-week exposure of LuP increased mutation frequencies and cotreatment with PCP suppressed these increases in both organs. Given that intact levels of SULT activity in the liver were much higher than in the kidneys of rodents, SULT1A may predominantly activate LuP in the liver, consequently leading to genotoxicity not only in the liver but also in the kidney. Thus, species differences should be considered in human risk assessment of renal carcinogens activated by SULT1A as in the case of the corresponding liver carcinogens.

Sulfotransferase-1A1-dependent bioactivation of aristolochic acid I and N-hydroxyaristolactam I in human cells.

Aristolochic acids (AA) are implicated in the development of chronic renal disease and upper urinary tract carcinoma in humans. Using in vitro approaches, we demonstrated that N-hydroxyaristolactams, metabolites derived from partial nitroreduction of AA, require sulfotransferase (SULT)-catalyzed conjugation with a sulfonyl group to form aristolactam-DNA adducts. Following up on this observation, bioactivation of AA-I and N-hydroxyaristolactam I (AL-I-NOH) was studied in human kidney (HK-2) and skin fibroblast (GM00637) cell lines. Pentachlorophenol, a known SULT inhibitor, significantly reduced cell death and aristolactam-DNA adduct levels in HK-2 cells following exposure to AA-I and AL-I-NOH, suggesting a role for Phase II metabolism in AA activation. A gene knockdown, siRNA approach was employed to establish the involvement of selected SULTs and nitroreductases in AA-I bioactivation. Silencing of SULT1A1 and PAPSS2 led to a significant decrease in aristolactam-DNA levels in both cell lines following exposure to AA-I, indicating the critical role for sulfonation in the activation of AA-I in vivo Since HK-2 cells proved relatively resistant to knockdown with siRNAs, gene silencing of xanthine oxidoreductase, cytochrome P450 oxidoreductase and NADPH:quinone oxidoreductase was conducted in GM00637 cells, showing a significant increase, decrease and no effect on aristolactam-DNA levels, respectively. In GM00637 cells exposed to AL-I-NOH, suppressing the SULT pathway led to a significant decrease in aristolactam-DNA formation, mirroring data obtained for AA-I. We conclude from these studies that SULT1A1 is involved in the bioactivation of AA-I through the sulfonation of AL-I-NOH, contributing significantly to the toxicities of AA observed in vivo.

Genotoxicity of 1-methylpyrene and 1-hydroxymethylpyrene in Chinese hamster V79-derived cells expressing both human CYP2E1 and SULT1A1.

1-Methylpyrene (1-MP) is a widespread pollutant that is carcinogenic in animals following metabolic activation. Previous studies have shown that benzylic hydroxylation of 1-MP, catalyzed by multiple CYP isoforms, gives rise to 1-hydroxymethylpyrene (1-HMP), which becomes bioreactive following further metabolism by various sulfotransferase (SULT) isoforms. However, the mutagenic and chromosome damaging effects of 1-MP and 1-HMP in mammalian cells have not been investigated. In this study a Chinese hamster V79-derived cell line expressing both human CYP2E1 and human SULT1A1 was used to investigate the ability of 1-MP and 1-HMP to induce cytotoxicity (using the CCK-8 assay), micronuclei and Hprt gene mutations. The role of each enzyme was investigated through co-exposure in the presence of an enzyme inhibitor. We found that at concentrations of 0.5-4 µM and 5-20 µM, under conditions where no reduction in cell viability/growth occurred, 1-HMP and 1-MP induced micronuclei in V79-hCYP2E1-hSULT1A1 cells in a concentration-dependent manner; however, both compounds were inactive in V79 cells. Similarly, they both caused an increase in Hprt mutant frequency in V79-hCYP2E1-hSULT1A1 cells in these concentration ranges, with 1-MP impairing cell viability/growth at 10 µM and above in the mutagenicity assay. The compounds were again both inactive in V79 cells. The effects of 1-HMP in V79-hCYP2E1-hSULT1A1 cells were blocked or reduced by addition of pentachlorophenol (PCP), a SULT1 inhibitor; the genotoxicity of 1-MP was significantly reduced by either 1-aminobenotrazole, a CYP2E1 inhibitor, or PCP. The results suggest that human CYP2E1 and SULT1A1 cooperate to activate 1-MP and cause genotoxicity in mammalian cells.

Suppression of sulfoconjugation reduces the protective effect of ortho-aminoazotoluene on hepatocarcinogenesis induced by diethylnitrosamine in mice.

The effects of ortho-aminoazotoluene on carcinogenic activity of diethylnitrosamine were studied in CBA and ICR mice. Injection of ortho-aminoazotoluene before and after diethylnitrosamine led to a significant reduction of its anticarcinogenic effect, judging from significantly lower level of liver tumors. Pentachlorophenol, inhibitor of sulfotransferase (catalyzing the terminal stage of ortho-aminoazotoluene metabolic activity), stimulated its carcinogenic effect on mouse liver. On the other hand, pentachlorophenol reduced the protective effect of ortho-aminoazotoluene on diethylnitrosamine-induced hepatocarcinogenesis in mice. Presumably, the carcinogenic and anticarcinogenic effects of ortho-aminoazotoluene were realized by its initial form or intermediate (non-sulfated) metabolites.

Mutagenic activation reduces carcinogenic activity of ortho-aminoazotoluene for mouse liver.

Pentachlorophenol (aromatic amine and azo stain metabolic stimulation inhibitor) reduced the hepatocarcinogenic activity of 4-aminoazobenzene and reduced that of ortho-aminoazotoluene in suckling mice. Both 4-aminoazobenzene and ortho-aminoazotoluene exhibited mutagenic activity in Ames' test in vitro on S. typhimurium TA 98 strain with activation with liver enzymes; this mutagenic activity was similarly suppressed by adding pentachlorophenol into activation medium. Induction of xenobiotic metabolism enzymes, stimulating the mutagenic activity of ortho-aminoazotoluene, suppressed its carcinogenic effect on mouse liver. Hence, ortho-aminotoluene (the initial compound), but not its mutagenic metabolites, was the direct active hepatocarcinogen for mice.

Potential use of nitrate reductase as a biomarker for the identification of active and dormant inhibitors of Mycobacterium tuberculosis in a THP1 infection model.

The development of a macrophage-based, antitubercular high-throughput screening system could expedite discovery programs for identifying novel inhibitors. In this study, the kinetics of nitrate reduction (NR) by Mycobacterium tuberculosis during growth in Thp1 macrophages was found to be almost parallel to viable bacilli count. NR in the culture medium containing 50 mM of nitrate was found to be optimum on the fifth day after infection with M. tuberculosis. The signal-to-noise (S/N) ratio and Z-factor obtained from this macrophage-based assay were 5.4 and 0.965, respectively, which confirms the robustness of the assay protocol. The protocol was further validated by using standard antitubercular inhibitors such as rifampicin, isoniazid, streptomycin, ethambutol, and pyrazinamide, added at their IC(90) value, on the day of infection. These inhibitors were not able to kill the bacilli when added to the culture on the fifth day after infection. Interestingly, pentachlorophenol and rifampicin killed the bacilli immediately after addition on the fifth day of infection. Altogether, this assay protocol using M. tuberculosis-infected Thp-1 macrophages provides a novel, cost-efficient, robust, and easy-to-perform screening platform for the identification of both active and hypoxic stage-specific inhibitors against tuberculosis.

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12.

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to 'graze' on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan "plaque" formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.

[Metabolism inhibition stimulates, metabolism activation inhibits cancerogenic activity of ortho-aminoazotoluene in mouse liver].

Pentachlorophenol, an inhibitor of metabolic activation of aminoazo dyes was administered to suckling mice prior to o-aminoazotoluene (OAT). It was followed by formation of numerous preneoplastic nodules and tumors in the lungs and liver. At the same time, 2,3,7,8-tetrachlorodibenzo-p-dioxine treatment decreased their number in the liver while slightly increasing them in the lung. A possible mechanism of aminoazo dye carcinogenicity is suggested.

[Effect of hepatocarcinogenicity of estragole on the glucocorticoid-mediated induction of liver-specific enzymes and the activity of the transcription factors FOXA and HNF4 in the liver of mouse and rat].

The carcinogenic effects of estragole in mice of the earlier unexplored strain ICR has been studied. It has been shown that there is a distinct correlation between the extent of inhibition of glucocorticoid-mediated induction of tyrosine aminotransferase and trypthophan oxygenase after acute administration of estragole and the frequency of liver tumors after estragole exposure. Estragole inhibits the induction of these enzymes only in female mice, but not in male mice and rats. DNA-binding activities of liver-enriched transcription factors were investigated on carcinogen-susceptible and -resistant animals. Estragole decreases the HNF4 (hepatic nuclear factor 4) and FOXA DNA-binding activities only in susceptible female mice, but not in nonsusceptible male mice and rats and does not influence the C/EBP and HNF1 activities. Pentachlorophenol, which prevents the hepatocarcinogenic effect of estragole, abolishes its inhibitory effect on tyrosine aminotransferase and trypthophan oxygenase glucocorticoid induction and restores the FOXA and HNF4 DNA-binding activities. The parallelism between the hepatocarcinogenic effects of estragole and the inhibition of FOXA and HNF4 DNA-binding activities serves as an additional argument for the involvement of these factors in the mechanisms of tumor suppression in the liver.

1H NMR to investigate metabolism and energy supply in rhesus macaque sperm.

Sperm ATP is derived primarily from either glycolysis or mitochondrial oxidative phosphorylation. In the present studies, (1)H NMR spectroscopy was used to characterize the metabolite profile in primate sperm treated either with alpha-chlorohydrin (ACH), a known inhibitor of sperm glycolysis or pentachlorophenol (PCP), an uncoupler of oxidative phosphorylation. Sperm were collected from monkeys in the fall and spring, washed and incubated with either the media control, ACH (0.5mM) or PCP (50 microM). Using principal components analysis, PC1 scores plot indicated that the greatest level of variance was found between fall and spring samples and not chemical-treated samples. However, PC4 scores plot did show a consistent effect of ACH treatment. From the PC1 loadings plot, metabolites contributing to the seasonal differences were higher levels of formate in the fall and higher levels of carnitine and acetylcarnitine in the spring as well as possible differences in lipoprotein content. The PC4 loadings plot indicated that ACH treatment decreased lactate and ATP consistent with inhibition of glycolysis. Carnitine also was decreased and acetylcarnitine increased although the latter was not statistically significant. With PCP-treated sperm, no difference between control and treated samples could be discerned suggesting either that primate sperm are insensitive to uncoupling agents or that glycolysis played the more important role in maintaining sperm ATP levels. Overall, NMR studies may prove useful in the development of metabolomic markers that signal sperm metabolic impairments and have the potential to provide useful biomarkers for reproductive health.

Sperm mitochondrial integrity is not required for hyperactivated motility, zona binding, or acrosome reaction in the rhesus macaque.

Whether the main energy source for sperm motility is from oxidative phosphorylation or glycolysis has been long-debated in the field of reproductive biology. Using the rhesus monkey as a model, we examined the role of glycolysis and oxidative phosphorylation in sperm function by using alpha-chlorohydrin (ACH), a glycolysis inhibitor, and pentachlorophenol (PCP), an oxidative phosphorylation uncoupler. Sperm treated with ACH showed no change in percentage of motile sperm, although sperm motion was impaired. The ACH-treated sperm did not display either hyperactivity- or hyperactivation-associated changes in protein tyrosine phosphorylation. When treated with PCP, sperm motion parameters were affected by the highest level of PCP (200 microM); however, PCP did not cause motility impairments even after chemical activation. Sperm treated with PCP were able to display hyperactivity and tyrosine phosphorylation after chemical activation. In contrast with motility measurements, treatment with either the glycolytic inhibitor or the oxidative phosphorylation inhibitor did not affect sperm-zona binding and zona-induced acrosome reaction. The results suggest glycolysis is essential to support sperm motility, hyperactivity, and protein tyrosine phosphorylation, while energy from oxidative phosphorylation is not necessary for hyperactivated sperm motility, tyrosine phosphorylation, sperm-zona binding, and acrosome reaction in the rhesus macaque.

Ca(2+)-dependent glycolysis activation mediates apoptotic ATP elevation in HeLa cells.

It was previously shown that cells die with increased cytosolic ATP after stimulation with apoptotic inducers including staurosporine (STS). To identify the source of apoptotic ATP elevation, we monitored, in real time, the cytosolic ATP level in luciferase-expressing HeLa cells. A mitochondrial uncoupler or a respiration chain inhibitor was found to decrease cytosolic ATP by about 50%. However, even when mitochondrial ATP synthesis was suppressed, STS induced a profound elevation of intracellular ATP. In contrast, the STS-induced ATP increase was prevented by any of three inhibitors of the glycolytic pathway: 2-deoxyglucose, iodoacetamide, and NaF. The STS effect strongly depended on intracellular calcium and was mimicked by a calcium ionophore. We conclude that Ca(2+)-dependent activation of anaerobic glycolysis, but not aerobic mitochondrial oxidative phosphorylation, is responsible for the STS-induced elevation of ATP in apoptotic HeLa cells.

Effects of copper-phenanthroline on pentachlorophenol-induced adaptation and cell death of Escherichia coli.

OBJECTIVE: To evaluate the effects of copper-phenanthroline (CuOP) on pentachlorophenol (PCP)-induced adaptation and cell death of Escherichia coli. METHODS: Bacterial growth and adaptation to PCP were monitored spectrophotometrically at 600 nm. Inactivation of bacterial cells was determined from colony count on agar dishes. Cellular ATP content and accumulation of PCP were assessed by chemiluminescence and HPLC analysis respectively. The formation of PCP-Cu-OP complex was shown by UV-visible spectra. RESULTS: Escherichia coli (E. coli) could adapt to PCP, a wood preservative and insecticide used in agriculture. The adaptation of E. coli to PCP prevented its death to the synergistic cytotoxicity of CuOP plus PCP and declined cellular accumulation and uncoupling of oxidative phosphorylation of PCP. Furthermore, CuOP and PCP neither produced reactive oxygen species (ROS) nor had a synergistic effect on uncoupling of oxidative phosphorylation in E. coli. The synergistic cytotoxicity of CuOP and PCP in E. coli might be due to the formation of lipophilic PCP-Cu-OP complex. CONCLUSION: Our data suggested that adaptation of E. coli to PCP decreased the synergistic effects of CuOP and PCP on prokaryotic cell death due to the formation of lipophilic PCP-Cu-OP complex, but it had no effect on the uncoupling of oxidative phosphorylation and production of reactive oxygen species in E. coli.

Induction and inhibition of oocyte maturation by EDCs in zebrafish.

BACKGROUND: Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen. METHODS: In this study, stimulatory and inhibitory effects of EDCs and natural steroids on oocyte maturation were examined in zebrafish. For effective agents, some details about the mechanism in induction or inhibition of maturation were examined. Possible groups of DES interacting with the MIH receptor are discussed based on relative potency of steroids to induce maturation. RESULTS: Among agents tested, tamoxifen (TAM) and its metabolite 4-hydroxytamoxifen (4-OHT) showed stimulatory activity similar to DES. The time courses of the change in germinal vesicle breakdown and an intracellular molecular event (the synthesis of cyclin B) induced by TAM were indistinguishable from those induced by MIH. In contrast, pentachlorophenol (PCP) had a potent inhibitory effect on MIH-induced oocyte maturation. PCP inhibited not only MIH-induced maturation but also DES- and TAM-induced maturation. Methoxychlor also inhibited maturation when oocytes were pre-treated with this agent. CONCLUSION: These results suggest that EDCs act as agonists or antagonists in the induction of oocyte maturation in fish.

Application of Alamar blue/5-carboxyfluorescein diacetate acetoxymethyl ester as a noninvasive cell viability assay in primary hepatocytes from rainbow trout.

We have adopted the application of two fluorescent indicator dyes to studying the viability of monolayers of primary rainbow trout hepatocytes. The two fluorescent dyes--Alamar blue, which indicates metabolic activity of a cell, and 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which is an indirect measure of cell membrane integrity-are noninvasive and can be monitored conveniently directly in multiwell plates. According to these dyes, L-15 culture medium supported hepatocyte viability over 96 h more stably than did M199. The two dyes proved to be capable of detecting a concentration-dependent toxic insult to hepatocytes caused by the model compound, pentachlorophenol. In contrast, a lack of impact on cell viability was indicated for up to 10(-5) M 17beta-estradiol, and that observation was supported by the induction of vitellogenin (VTG) mRNA/protein as indicator of hepatocyte-differentiated function. Application of the Alamar blue/CFDA-AM for 30 min did not alter gene expression either specifically as reflected by VTG or generally as reflected by a random selection of gene sequences that were amplified by differential display reverse transcription PCR (dd-rt-PCR). Thus, the assay represents a resource-efficient way of integrating measures of cell viability and gene expression that should aid in the interpretation of in vitro results. The assay can be applied repeatedly to the same set of cells and can be performed just prior to analysis of gene expression.

Induction of cytotoxicity, aldehydic DNA lesions, and poly(ADP-ribose) polymerase-1 activation by catechol derivatives of pentachlorophenol in calf thymus DNA and in human breast cancer cells.

The purpose of this study was to investigate the degree of chlorination of catechol (CAT) derivatives of pentachlorophenol (PCP) on the induction of cytotoxicity and DNA damaging effects in calf thymus DNA (ct-DNA) and in two human breast carcinoma cell lines. Results indicated that with the addition of the transition metal copper(II), increases in the amount of aldehydic DNA lesions (ADL) were detected in ct-DNA exposed to PCP-derived CATs over the corresponding control. The DNA lesions induced by various degrees of chlorination of PCP-derived CATs decrease in the rank order CAT congruent with 4-chlorocatechol (4-ClCAT) > 4,5-dichlorocatechol (4,5-Cl2CAT) > 3,4,5-trichlorocatechol (3,4,5-Cl3CAT) > tetrachlorocatechol (Cl4CAT). In contrast, Cl4CAT was the only congeneric form of PCP-derived catechols that induced a significant increase in the number of ADL in human MCF-7 cells, and this only occurred when glutathione was depleted. Pretreatment with copper(I) and iron(II) chelators significantly reduced the formation of ADL in cells exposed to Cl4CAT. The data also indicated that the ADL induced by Cl4CAT in MCF-7 cells contain approximately 70% putrescine excisable ADL. This evidence confirmed that the ADL induced by Cl4CAT in MCF-7 cells were derived from oxidative events. In addition, we demonstrated that the depletion of NAD(P)H in human T47D cells exposed to chlorinated CATs decreased in the rank order Cl4CAT >> 4-ClCAT congruent with CAT. The depletion of NAD(P)H induced by Cl4CAT in T47D cells was partially blocked by catalase, superoxide dismutase, dimethyl sulfoxide, and copper(I) and iron(II) specific chelators. Additionally, the depletion of NAD(P)H in T47D cells exposed to Cl4CAT (1-10 microM) was completely blocked by three types of poly(ADP-ribose) polymerase-1 inhibitors. This evidence suggests that Cl4CAT induces an imbalance in DNA repair and the subsequent accumulation of DNA strand breaks in human cultured cells. Overall, these findings indicate that dechlorination may decrease the potentials of chlorinated catechols to induce oxidative DNA lesions and cytotoxic effects in living cells.

Pentachlorophenol reduces B lymphocyte function through proinflammatory cytokines in Carassius auratus.

Cytokines and IgM levels are important parameters in the acquired immunity of fish. In the present study, the effect of pentachlorophenol (PCP) on the expression of proinflammatory cytokines, TNF-alpha (tumor necrosis factor-alpha) and IL-1beta (interleukin 1) mRNA levels of crucian carp, were determined by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. To put the deduction of PCP's immunotoxicity on B cell function, B cells secretion of IgM under exposure of PCP-administrated fish macrophage was determined by enzyme-linked immunosorbent assay (ELISA), and the ability of B cells to secrete IgM was determined by enzyme-linked immunospot (ELISPOT). The results showed that the mRNA expressions of these two cytokines were suppressed by the administration of PCP. The supernatants from PCP-administrated fish macrophage showed less stimulation on B cell, lower maturation of B cells and secretion of IgM. These results suggested that PCP might have impact on micromilieu immune factors as proinflammatory cytokines.

Anti-estrogenic activity of fifty chemicals evaluated by in vitro assays.

We examined the anti-estrogenic activity of 50 chemicals by the yeast two-hybrid assay and detected the activity of hexachlorophene, pentachlorophenol, and vitamin K3 (menadione), in that order. These chemicals were also observed to inhibit the transcriptional activity of 17beta-estradiol in a reporter gene assay system using MCF-7 cells, estrogen receptor-positive breast cancer cells, and to bind directly to estrogen receptor alpha in a competitive binding assay system, although the order of the activity was slightly different among the 3 assays. These findings suggested that three of fifty chemicals could inhibit estrogen activity by competitive binding with 17beta-estradiol to the estrogen receptor.