Effects of tebuconazole on cytochrome P450 enzymes, oxidative stress, and endocrine disruption in male rats.

The major objective of the present study was to determine the ability of a triazole fungicide tebuconazole to induce cytochrome P450-dependent monooxygenases, oxidative stress, and endocrine-disrupting activity using male rats treated with tebuconazole at 10, 25, and 50 mg/kg p.o. once daily for 28 days. In liver, tebuconazole dose-dependently increased microsomal contents of cytochrome P450 and cytochrome b5 and the activities of NADPH-cytochrome P450 reductase, 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, pentoxyresorufin O-dealkylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase. In kidney, tebuconazole increased 7-ethoxycoumarin O-deethylase activity without affecting other monooxygenase activities. In marked contrast to liver and kidney, tebuconazole decreased testicular 7-ethoxyresorufin O-deethylase, methoxyresorufin O-demethylase, 7-ethoxycoumarin O-deethylase, aniline hydroxylase, and erythromycin N-demethylase activities. The results of immunoblot analysis of liver microsomes of controls and tebuconazole-treated rats revealed that tebuconazole induced CYP1A1/2, CYP2B1/2, CYP2E1, and CYP3A proteins in liver. Additions of tebuconazole to liver microsomes inhibited microsomal 7-ethoxycoumarin O-deethylase activity in vitro (IC50 = 1.50-1.69 µM). Treatment of rats with tebuconazole decreased glutathione content and increased glutathione S-transferase, superoxide dismutase, catalase, and glutathione peroxidase activities in liver; increased superoxide dismutase activities in kidney and testis; but decreased glutathione S-transferase activity in testis. Treatments with tebuconazole decreased serum testosterone concentration and cauda epididymal sperm count. The present study demonstrates that tebuconazole induces a multiplicity of CYPs and oxidative stress in liver; inhibits testicular P450 and glutathione S-transferase activities; and produces anti-androgenic effects in male rats.

Induction of cardiac fibrosis and transforming growth factor-ß1 by motorcycle exhaust in rats.

Motorcycle exhaust (ME) is a major source of air pollution and a potential health hazard in urban areas where motorcycles are a popular means of transportation. The main objectives of this study were to determine the ability of ME to cause cardiotoxicity in rats and investigate the possible mechanisms of toxicity. Male rats were exposed to 1:10 diluted ME by inhalation 2 h daily and Monday through Friday for 8 weeks. Exposure to ME increased heart weight and decreased cardiac antioxidant enzymes glutathione S-transferase (GST), superoxide dismutase and glutathione peroxidase activities in a concentration- and time-dependent manner. Analysis of echocardiographic parameters indicated that ME increased left ventricle posterior wall thickness, interventricular septum thickness and left ventricle mass. Histopathological examinations of the hearts revealed that ME exposure caused focal cardial degeneration and necrosis, mononuclear cell infiltration, and fibrosis. The results of reverse transcriptase-polymerase chain reaction studies showed that ME decreased GST-M1 and GST-P1 mRNA expression and increased the expression of proinflammatory cytokine interleukin-1ß, hypertrophy marker atrial natriuretic peptide, fibrosis markers type I and III collagen, profibrotic cytokine connective tissue growth factor, and hypertrophy and fibrosis mediator transforming growth factor (TGF)-ß1 in the heart. The data of Western blot analysis showed that cardiac TGF-ß1 protein was induced by ME. These findings demonstrate that subchronic ME exposure caused hypertrophy and fibrosis, and modulated GST and TGF-ß1 expression in rat heart possibly by mechanisms involving oxidative stress and inflammation.

Factors from human embryonic stem cell-derived fibroblast-like cells promote topology-dependent hepatic differentiation in primate embryonic and induced pluripotent stem cells.

The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

Potential roles of fibroblast growth factor-9 in the benzo(a)pyrene-induced invasion in vitro and the metastasis of human lung adenocarcinoma.

Fibroblast growth factor (FGF)-9 belongs to the FGF family which modulate cell proliferation, differentiation, and motility. Benzo(a)pyrene is a polycyclic aromatic hydrocarbon (PAH) and ubiquitous environmental carcinogen present in automobile exhaust, cigarette smoke, and foods. The major purposes of this study were to explore the roles of FGF-9 in the benzo(a)pyrene-induced lung cancer invasion in vitro and the metastatic development of lung adenocarcinoma in human. The data of RT-PCR analysis indicated that treatments of human lung adenocarcinoma CL5 cells with benzo(a)pyrene and a PAH mixture motorcycle exhaust particulate (MEP) extracts increased FGF-9 mRNA expression. The increased expression was blocked by cotreatments with a p38 mitogen-activated protein kinase inhibitor SB202190 and an extracellular signal-regulated kinase inhibitor PD98059. The results of immunoblot analysis and Matrigel assay showed that benzo(a)pyrene and MEP extracts produced a concomitant induction of FGF-9 protein and invasive ability of CL5 cells. The benzo(a)pyrene- and MEP-induced invasion was suppressed by FGF-9 neutralizing antibodies. The results of immunohistochemistry analysis of human lung adenocarcinoma specimens showed that FGF-9 protein was detected in the adenocarcinoma cells but not in normal epithelium. FGF-9 staining intensity was positively correlated with status of disease and degree of lymph node metastasis in these lung adenocarcinomas. These present findings suggest that FGF-9 has potential roles in benzo(a)pyrene-induced CL5 cell invasion and human lung adenocarcinoma metastasis.

Induction of CYP1A1, 2B, 2E1 and 3A in rat liver by organochlorine pesticide dicofol.

The present study has determined the ability of dicofol, an organochlorine pesticide, to induce cytochrome P450 using rats treated with 1, 10, and 25mg/kg dicofol intraperitoneally for 4 days. Treatments with 10 and 25mg/kg dicofol produced dose-related increases of cytochrome P450 and cytochrome b(5) contents and NADPH-cytochrome c reductase, 7-ethoxyresorufin O-deethylase, pentoxyresorufin O-dealkylase, aniline hydroxylase, and erythromycin N-demethylase activities in liver microsomes. The treatments also increased glutathione S-transferase and superoxide dismutase activities in liver cytosol. Dicofol at 1mg/kg produced a general trend towards increases of the aforementioned enzyme levels. The results of immunoblot analyses showed that 10 and 25mg/kg dicofol increased protein levels of CYP1A1, CYP2B, CYP2E1, and 3A in liver. RT-PCR data indicated that dicofol induced mRNA expression of liver CYP1A1, CYP2B, and CYP3A. Pretreatments of rats with 10 and 25mg/kg dicofol decreased phenobarbital-induced sleeping time by 34% and 39%, respectively. Dicofol pretreatment at 25mg/kg increased CCl4-induced serum alanine aminotransferase activity by 4.3-fold and aspartate aminotransferase activity by 4.1-fold. The present study demonstrates that dicofol has the ability to induce CYP1A1, CYP2B, CYP2E1, and CYP3A in the liver and increase phenobarbital metabolism and CCl4 toxicity in rats.

Reduced expression of von Hippel-Lindau gene in subjects exposed to polychlorinated biphenyls and dibenzofurans.

Polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PCDFs) are ubiquitous pollutants found in the environment and human tissues. A cohort in Taiwan has undergone follow-up for 24 years after exposure to high levels of PCBs and PCDFs. The incidence of chloracne, hyperkeratosis, and abnormal nail was increased among exposed people. We conducted a study to identify the genes whose expressions were affected by such exposure. A cDNA microarray system consisting of 908 genes was used for pooled serum samples from non-smoking men exposed to PCBs and PCDFs (n=15) and their matched referents (n=15) in triplicate. After adjusting for background and housekeeping genes, genes with different expressions between the exposure and reference groups were determined by both regression and cluster analysis, and further confirmed by real-time RT-PCR. The tumor suppressor gene von Hippel-Lindau (VHL) was found to be down-regulated in the microarray analysis. VHL gene expression levels were also found to be positively associated with age, shown by real-time RT-PCR. Upon age adjustment, VHL gene expression was reduced in Yucheng ("oil disease") subjects as compared to referents. Among Yucheng people, those with abnormal nails had lower VHL expressions than those without abnormal nails. These findings provide new insights into the potential role of VHL in health conditions associated with PCB and PCDF exposures.

Induction of hepatic glutathione S-transferase and UDP-glucuronosyltransferase activities by ketamine in rats.

BACKGROUND: Ketamine has been shown to induce rat cytochrome P-450 in a way similar to phenobarbital. However, whether ketamine is able to induce glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UGT), two major phase II drug-metabolizing enzymes, remains unclear. The present study aimed to investigate the effect of ketamine on GST and UGT activities in rats. METHODS: In a dose-response study, male adult Wistar rats were treated with 10, 20, 40 or 80 mg/kg ketamine intraperitoneally twice daily for 4 days. Livers were removed 1 day after ketamine treatment and hepatic GST and UGT activities were determined. In a reversibility study, rats were treated with 80 mg/kg ketamine intraperitoneally twice daily for 4 days and killed 1, 2, 3 or 4 days after the last dose of ketamine. Livers were removed and hepatic GST and UGT activities were determined. RESULTS: The results of the dose-response study showed that treatment of rats with 10, 20, 40, or 80 mg/kg ketamine produced 19%, 20%, 18%, and 25% increases respectively in the catalytic activity of hepatic cytosolic GST, and 41%, 41%, 35%, and 38% increases respectively in the catalytic activity of microsomal UGT. The results of the reversibility study showed that the GST activities of the rats killed 1, 2, 3, or 4 days after ketamine treatment were 62%, 88%, 46% and 65% higher than the activity of the control group. The UGT activities of the rats killed 1, 2, 3, or 4 days after ketamine treatment were 56%, 53%, 54% and 72% higher than the activity of the control group. CONCLUSION: Ketamine is able to induce the activities of hepatic GST and UGT in rats. The induced GST and UGT activities persist for at least 4 days after cessation of ketamine. The results suggest the possibility of interactions of drugs related to phase II enzyme induction in chronic ketamine users.

Modulation of serum concentrations and hepatic metabolism of 17beta-estradiol and testosterone by amitraz in rats.

The present study has investigated the ability of amitraz, a widely used formamidine pesticide, to modulate serum concentrations and liver microsomal metabolism of 17beta-estradiol (E2) and testosterone in rats. Amitraz was administered intraperitoneally to male rats for 4 days and to intact female rats or ovariectomized (OVX) and 0.5 mg/kg E2-supplemented female rats for 7 days. E2 and metabolites were analyzed by gas chromatography-electron capture detection and testosterone and metabolites were analyzed by high-pressure liquid chromatography. In OVX and E2-supplemented females, 50 mg/kg amitraz caused an 85% decrease of serum E2 concentration and a marked increase of 2-OH-E2 concentration. Amitraz at 25 and 50 mg/kg produced 9.0-fold or greater increases of serum testosterone and 2beta-OH-testosterone levels in males. Amitraz at 25 mg/kg resulted in no or minimal increases of liver microsomal formation of E2 or testosterone metabolites. Amitraz at 50 mg/kg produced 1.4- to 3.6-fold increases of 2-OH-E2; estrone; 2beta-, 6beta-, and 16alpha-OH-testosterone; and androstenedione formation in males and intact females. Amitraz at 50 mg/kg preferentially increased intact female 16beta-OH-testosterone production by 8.6-fold. In OVX females, E2 supplement alone or cotreatment with E2 and 50 mg/kg amitraz produced 1.3- to several-fold increases of 2- and 4-OH-E2 formation and 2beta- and 16alpha-OH-testosterone production. The cotreatment increased 6beta- and 16beta-OH-testosterone formation by 1.8- and 1.6-fold, respectively. The present findings show that amitraz induces hepatic E2 and testosterone metabolism in male and female rats, decreases serum E2 concentration in OVX and E2-supplemented females, but increases serum testosterone in males.

Motorcycle exhaust induces reproductive toxicity and testicular interleukin-6 in male rats.

Motorcycle exhaust (ME) from two-stroke engines contains many toxicants and poses a potential health hazard. The major objectives of the present study were to investigate the male reproductive toxicity of ME and the underlying mechanisms of toxicity. Male Wistar rats were exposed to ME by inhalation 1 h each in the morning and afternoon, Monday through Friday. Exposures to 1:50 diluted ME for 4 weeks or to 1:10 diluted ME for 2 and 4 weeks showed concentration- and time-dependent decreases of testicular weight, spermatid number, and cauda epididymal sperm number. Subsequent studies were done using 4-week exposure to 1:10 diluted ME. ME caused histopathological changes including testicular spermatocytic necrosis and seminiferous tubule atrophy and cauda epididymal formation of clusters of pyknotic and necrotic sperm cells. ME-exposed male rats mated with untreated females showed decreases of male mating index and female fertility index and an increase of implantation site loss. ME decreased 7-ethoxycoumarin O-deethylase and superoxide dismutase activities but induced proinflammatory cytokine interleukin-6 (IL-6) messenger RNA (mRNA) in the testis. Male rats were exposed to ME with or without cotreatment with 50 mg/kg vitamin E orally for 4 weeks. ME decreased serum testosterone concentration. This effect was reversed by cotreatment with vitamin E. ME decreased testicular spermatid number and induced IL-6 mRNA and protein. These effects were also reversed by the vitamin E cotreatment. The present findings show that ME causes male reproductive effects and induces testicular IL-6 in rats by mechanisms involving induction of oxidative stress and inhibition of steroidogenesis.

Upregulation of cyclooxygenase-2 by motorcycle exhaust particulate-induced reactive oxygen species enhances rat vascular smooth muscle cell proliferation.

Long-term exposure to particulate air pollution has been implicated as a risk factor for cardiovascular disease and mortality. Short-term exposure has also been suggested to contribute to complications of atherosclerosis. Aberrant regulation of smooth muscle cell proliferation is thought to associate with the pathophysiology of vascular disorders such as atherosclerosis. In this study, we investigate the influence of organic extracts of motorcycle exhaust particulates (MEPE) on rat vascular smooth muscle cell (VSMC) proliferation and related regulation signaling. Exposure of VSMCs to MEPE (10-100 microg/mL) enhanced serum-induced VSMC proliferation. The expression of proliferating cell nuclear antigen (PCNA) was also enhanced in the presence of MEPE. VSMCs treated with MEPE induced the increase in the extent of cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E 2 production, whereas the level of COX-1 protein was unchanged. Moreover, MEPE increased the production of reactive oxygen species (ROS) in VSMCs in a dose-dependent manner. MEPE could also trigger time-dependently extracellular signal-regulated kinase (ERK)1/2 phosphorylation in VSMCs, which was attenuated by antioxidants N-acetylcysteine (NAC) and pyrrolidinedithiocarbamate (PDTC). The level of translocation of nuclear factor (NF)-kappaB-p65 in the nuclei of VSMCs was also increased under MEPE exposure. The potentiating effect of MEPE on serum-induced VSMC proliferation could be abolished by COX-2 selective inhibitor NS-398, specific ERK inhibitor PD98059, and antioxidants NAC and PDTC. Taken together, these findings suggest that MEPE may contribute to the enhancement of the pathogenesis of cardiovascular diseases by augmenting proliferation of VSMCs through a ROS-regulated ERK1/2-activated COX-2 signaling pathway.

Induction and inhibition of cytochrome P450-dependent monooxygenases of rats by fungicide bitertanol.

The effects of fungicide bitertanol on cytochrome P450-dependent monooxygenases were studied using rats treated intraperitoneally with the N-substituted triazole for 4 days. Treatment with 10, 25, and 100 mg/kg bitertanol produced 2-, 4-, and 14-fold increases of 7-ethoxyresorufin O-deethylation activity in liver microsomes, respectively. Immunoblot analysis of microsomal proteins revealed that 25 mg/kg bitertanol increased CYP1A1 protein in the liver, kidney, and lung by 10-, 13-, and 17-fold, respectively. Bitertanol produced smaller increases of CYP2B and CYP3A catalytic activity and protein than that of CYP1A1 in liver. RT-PCR analysis of total RNA indicated that bitertanol-induced CYP1A1, CYP2B, and CYP3A mRNA. Additions of 0.01-100 microM bitertanol to liver microsomes from rats treated with 25 mg/kg bitertanol or 3-methylcholanthrene inhibited microsomal 7-ethoxyresorufin O-deethylation activity (IC(50)=0.8 or 0.9 microM). Bitertanol at 100 mg/kg increased liver UDP-glucuronosyltransferase and glutathione S-transferase activities by 2-fold. Bitertanol at 25 mg/kg produced a minor increase in metabolic activation of benzo[a]pyrene by liver S-9 fraction in the Ames mutagenicity test while the increase was blocked by addition of 100 microM bitertanol. These findings show that bitertanol is an inducer of CYP1A1, CYP2B, and CYP3A in vivo and an inhibitor of CYP1A catalytic activity in vitro.

CYP2C9 polymorphism and warfarin sensitivity in Taiwan Chinese.

BACKGROUND: Warfarin prevents thromboembolism in patients with prosthetic heart valvular replacement. Cytochrome P4502C9 (CYP2C9) is polymorphic in human and is principally responsible for the metabolism of warfarin. However, known CYP2C9 polymorphisms cannot entirely account for the low dose requirement of warfarin in Chinese-Taiwanese receiving mitral valve replacement. We screened a new polymorphism of CYP2C9 and investigated its role in warfarin sensitivity. METHODS: We examined warfarin dose requirements in 239 Chinese-Taiwanese patients who had attended a cardiac surgery clinic in National Taiwan University Hospital. DNA samples were obtained from 106 Chinese-Taiwanese (37 patients and 69 unrelated healthy controls), and healthy control subjects of Caucasians (n=28) and African-Americans (n=28). Four out of those 37 patients were poor metabolizers of warfarin, and their DNA were subjected to sequencing analysis. Moreover, CYP2C9 genotyping analyses were performed using PCR-RFLP analysis. The chi2 test and Fisher's exact test were used to compare the differences of the allelic frequency and genotype. The association between warfarin dose requirement and genetic polymorphism of CYP2C9 was also analysed. RESULTS: The mean daily warfarin dose was 3.11+/-1.62 mg for the maintenance of the international normalized ratio of 2 to 3 in 239 patients. A single nucleotide substitution from G to C was found in this study. This SNP, G-65/C, is in intron 3, 65 base pairs upstream of exon 4. The allelic frequencies of C-65 in healthy controls were 0.125, 0.058 and approximately 0 with respect to African-American, Chinese-Taiwanese and Caucasian, implying inter-ethnic variations of the C-65 allele. In addition, patients who were carrier of either the heterozygous or homozygous C-65 variant received half of the usual warfarin dose. CONCLUSION: The novel intronic G-65/C mutation appears to be inter-racially different in allelic frequency, and that the anticoagulation was affected in response to warfarin sensitivity in Chinese-Taiwanese patients receiving mitral valve replacement.

The abortifacient effects from the seeds of Coix lachryma-jobi L. var. ma-yuen Stapf.

This study evaluated the abortifacient effects of the extracts of seeds of Coix lachryma-jobi L. var. ma-yuen Stapf (adlay) in pregnant rats. Pregnant rats were treated with oral administration of adlay seed extracts on d 6 of pregnancy and their fetuses were examined for growth and malformations on d 20 of pregnancy. Following oral administration of 1 g/kg body weight of water extracts but not methanolic extracts, fetal resorptions were significantly increased and mortality of postimplantation was increased. There were no significant differences in the uterine and fetal weight compared to control. Fetal malformations were not observed in the adlay seed extracts-treated pregnant rats. The contractile activity of uteri isolated from rats on d 20 of pregnancy was assessed. The spontaneous uterine contractions were significantly enhanced in rats treated with water extracts of adlay seeds (1 g/kg body weight). Immunoblotting of uteri from rats treated with water extracts of adlay seeds demonstrated an induction of cyclooxygenase-2 (COX-2) protein expression. The water extracts of adlay seeds also enhanced extracellular signal-regulated protein kinase (ERK) 1/2 phosphorylation and protein kinase C (PKC)-alpha translocation from cytosolic to particulate fractions in uteri. These results indicate that the water extracts of adlay seeds are capable of inducing embryotoxicity and enhancing uterine contractility during pregnancy. The enhanced activities of PKC-alpha, ERK1/2, and COX-2 may contribute to these responses.

Induction of rat hepatic cytochrome P-450 by ketamine and its toxicological implications.

Ketamine is a common intravenous anesthetic and a frequent drug of abuse, alone or in combination with cocaine. However, the pharmacokinetic effects of ketamine have not been fully investigated. This study determined the effects of ketamine on cytochrome P-450 (P-450)-dependent catalytic activities, protein levels, and hepatotoxicity using male Wistar rats treated with 10, 20, 40, or 80 mg/kg ketamine intraperitoneally twice daily for 4 d. Treatment with ketamine produced a dose-dependent increase of pentoxyresorufin O-dealkylation activity of liver microsomes. Treatment with 80 mg/kg ketamine resulted in 14-, 3-, and 2-fold rise in O-dealkylation of pentoxyresorufin, ethoxyresorufin, and methoxyresorufin of rat liver microsomes, respectively. The treatment produced 31% and 86% increases in 7-ethoxycoumarin O-deethylation and erythromycin N-demethylation, respectively. In addition, aniline hydroxylation activity was elevated by 62%. Protein blot analysis of liver microsomal proteins revealed that 80 mg/kg ketamine induced P-450 1A, 2B, 2E1, and 3A proteins by 2-, 13-, 2-, and 2-fold, respectively. In reversibility study, ketamine-induced pentoxyresorufin O-dealkylation, 7-ethoxycoumarin O-deethylation, erythromycin N-demethylation, and methoxyresorufin O-demethylation activities of liver microsomes prepared from rats 4 d after ketamine treatment were 75%, 48%, 29%, and 38% lower than the respective activities of liver microsomes prepared from rats 1 d after treatment. Protein blot analysis showed that ketamine-induced P-450 2B1/2 proteins also decreased in a time-dependent manner in 4 d. In hepatotoxicity study, treatment of rats with 1 ml/kg CCl4 produced a 7-fold increase in serum alanine aminotransferase activity level and a 17-fold rise in rats pretreated with 80 mg/kg ketamine for 4 d. Treatment of ICR mice with 120 mg/kg cocaine produced a 17% mortality, whereas the same dose of cocaine produced a 50% mortality in mice pretreated with ketamine. Treatment of mice with 100 mg/kg cocaine produced a 76-fold increase in serum alanine aminotransferase activity level and a 260-fold rise in mice pretreated with 80 mg/kg ketamine for 4 d. The present study shows that ketamine induces the expression of multiple forms of P-450 in rat liver microsomes and increases CCl4-induced liver toxicity and cocaine-mediated acute toxicity. Other potential pharmacological or toxicological events related to ketamine use need to be further explored.

Induction of fibroblast growth factor-9 and interleukin-1alpha gene expression by motorcycle exhaust particulate extracts and benzo(a)pyrene in human lung adenocarcinoma cells.

Motorcycle exhaust particulates (MEP) contain carcinogenic polycyclic aromatic hydrocarbons including benzo(a)pyrene. This study has determined the ability of MEP to alter the expression of select genes from drug metabolism, cytokine, oncogene, tumor suppressor, and estrogen signaling families of human lung adenocarcinoma CL5 cells. cDNA microarray analyses and confirmation studies were performed using CL5 cells treated with 100 microg/ml MEP extract for 6 h. The results showed that MEP increased the mRNA levels of metabolic enzymes CYP1A1 and CYP1B1, proinflammatory cytokines interleukin (IL)-1alpha, IL-6, and IL-11, fibroblast growth factor (FGF)-6 and FGF-9, vascular endothelial growth factor (VEGF)-D, oncogene fra-1, and tumor suppressor p21. In contrast, MEP decreased tumor suppressor Rb mRNA in CL5 lung epithelial cells. Treatment with 10 microM benzo(a)pyrene for 6 h altered gene expression profiles, in a manner similar to those by MEP. Induction of IL-1alpha, IL-6, IL-11, and FGF-9 mRNA by MEP and benzo(a)pyrene was concentration and time dependent. Cotreatment with 2 mM N-acetylcysteine blocked the MEP- and benzo(a)pyrene-mediated induction. Treatment with MEP or benzo(a)pyrene increased IL-6 and IL-11 releases to CL5 cell medium. Incubation of human lung fibroblast WI-38 with MEP- or benzo(a)pyrene-induced CL5 conditioned medium for 4 days stimulated cell growth of the fibroblasts. Inhalation exposure of rats to 1:10 diluted motorcycle exhaust 2 h daily for 4 weeks increased CYP1A1, FGF-9, and IL-1alpha mRNA in lung. This present study shows that MEP and benzo(a)pyrene can induce metabolic enzyme, inflammatory cytokine, and growth factor gene expression in CL5 cells and stimulate lung epithelium-fibroblast interaction.

Effects of amitraz on cytochrome P450-dependent monooxygenases and estrogenic activity in MCF-7 human breast cancer cells and immature female rats.

This study investigated the ability of amitraz, a formamidine insecticide, to induce cytochrome P450-dependent monooxygenases and to disrupt estrogenic activity in human breast cancer MCF-7 cells and immature female rats. In MCF-7 cells, treatment with 10 microM amitraz for 24 h increased 7-ethoxyresorufin O-deethylase activity in cell homogenate. Treatment of MCF-7 cells with 1 and 10 microM amitraz for 3 h replaced previously bound [(3)H]17beta-estradiol (E(2)) from estrogen receptors. Treatment with 0.1 and 1 microM amitraz for 2 days inhibited [(3)H]thymidine incorporation into the DNA of MCF-7 cells while the inhibition was blocked in cells co-treated with 1 nM E(2) and amitraz. In immature female rats, treatment with 50 mg/kg amitraz intraperitoneally for 3 days increased cytochrome P450 content, 7-ethoxyresorufin, methoxyresorufin and pentoxyresorufin O-dealkylases, and benzo[a]pyrene hydroxylase activities in liver microsomes. The results of immunoblot analysis revealed that amitraz induced liver microsomal CYP1A1/2, 2B1/2B2, and 3A proteins. Treatment with 10 and 25 mg/kg amitraz for 3 days dose-dependently decreased uterine weight and peroxidase activity in immature female rats while the decreases were blocked in rats co-treated with 10 microg/kg E(2) and 10 or 25 mg/kg amitraz. These in vitro and in vivo findings suggest that amitraz induces multiple forms of P450 and exerts weak antiestrogenic activity.

Endocrine-disrupting activity in carbendazim-induced reproductive and developmental toxicity in rats.

This study was designed to investigate the endocrine-disrupting activity of carbendazim-induced reproductive and developmental toxicity in Sprague-Dawley rats treated orally with the fungicide. Cotreatment of male rats with 675 mg/kg carbendazim and 50 or 100 mg/kg flutamide, an androgen receptor antagonist, once daily for 28 d blocked decrease of testis weight induced by treatment with carbendazim alone. The cotreatment prevented losses of spermatozoa and cell morphology and decrease of sperm concentration induced by carbendazim. Premating treatment of male and female rats with 200 mg/kg carbendazim for 28 d produced androgenic effects including incomplete development of uterine horn, enlargement of uretha, absence of vagina, and induction of seminal vesicles in female offspring, without marked effects in male offspring. Premating treatment with 100mg/kg benomyl, the parent compound of carbendazim, resulted in incomplete development of uterine horn and absence of vagina in female offspring and produced testis and epidydimis atropy in male offspring. Treatment of male rats with 25, 50, 100, 200, 400, and 800 mg/kg carbendazim for 56 d produced dose-dependent increases of androgen receptor concentrations in testis and epididymis. Additions of 5, 50, and 500 microM carbendazim to testis extract from untreated rats replaced binding of [3H]-5 alpha-dihydrotestosterone to androgen receptor in a concentration-dependent manner. The present study demonstrates that reproductive toxicity induced by carbendazim is blocked by an androgen receptor antagonist in male rats and developmental toxicity of the fungicide shows androgenic properties in female offspring. These results suggest that androgen- and androgen receptor-dependent mechanisms are possibly involved in carbendazim-induced toxicity.

Antiestrogenic effects of motorcycle exhaust particulate in MCF-7 human breast cancer cells and immature female rats.

The emissions from 2- and 4-stroke motorcycles pollute the air of urban areas where motorcycle is a popular means of transportation. This study aimed to determine the endocrine-disrupting activity of motorcycle exhaust particulate (MEP) using MCF-7 human breast cancer cells and immature female Wistar rats treated with organic extracts of MEP. Treatments with 1, 10, and 50 microg/ml MEP extract for 2 and 4 days produced dose-dependent inhibition of thymidine incorporation and cell growth, respectively, in untreated and 1 nM 17beta-estradiol (E2)-treated cells. Treatments of MCF-7 cells with MEP extract replaced [3H]E2 from the estrogen receptor in a time- and concentration-dependent manner. These antiestrogenic and receptor binding properties of MEP extract were blocked by cotreatment of the cells with 2 microM alpha-naphthoflavone, a cytochrome P450 inhibitor and aryl hydrocarbon receptor antagonist. E2 metabolism and HPLC analysis showed that treatment of MCF-7 cells with 50 microg/ml MEP extract for 24 h increased E2 2- and 4-hydroxylation in microsomes. The MEP-mediated increase in E2 2-hydroxylation was inhibited by the addition of 1 microM alpha-naphthoflavone to MCF-7 microsomes. Cotreatment of immature female rats with 10 microg/kg E2 and 10 mg/kg MEP extract intraperitoneally for 3 days decreased the E2-induced uterine weights. MEP extract alone showed no effect on rat uterine weight. The endocrine-disrupting activity of MEP extract was further confirmed in parallel experiments using MCF-7 cells and immature female rats treated with benzo(a)pyrene, an MEP constituent compound. The present findings demonstrate that MEP extract is antiestrogenic in vitro and in vivo and cytochrome P450 induction is an underlying mechanism.

Effects of motorcycle exhaust inhalation exposure on cytochrome P-450 2B1, antioxidant enzymes, and lipid peroxidation in rat liver and lung.

The effects of motorcycle exhaust (ME) on metabolic and antioxidant enzymes and lipid peroxidation were determined using male rats exposed to 1:10 diluted ME by inhalation 2 h daily for 4 wk. For microsomal cytochrome P-450 enzymes, ME resulted in threefold increases of 7-ethoxyresorufin and pentoxyresorufin O-deethylase activities in liver and a sixfold increase of 7-ethoxyresorufin O-deethylase activity and an 80% decrease of pentoxyresorufin O-dealkylase activity in lung. The results of immunoblot analysis of microsomal proteins revealed that ME increased liver and lung cytochrome P-450 1A1 with minimal effects on cytochrome P-450 2E1. ME increased cytochrome P-450 2B1/2 proteins in liver but decreased cytochrome P-450 2B1 in lung. ME did not change microsomal cytochrome P-450 enzyme activity or protein level in kidney. For phase II enzymes, ME resulted in 53% and twofold increases of cytosolic NAD(P)H:quinone oxidoreductase activities in liver and lung, respectively, and no effect on microsomal UDP-glucuronosyltransferase activities. For antioxidant enzymes, ME produced 23% and 35% decreases of superoxide dismutase, 9% and 27% decreases of catalase, and no changes of glutathione peroxidase activities in liver and lung cytosols, respectively. For lipid peroxidation, the results of thiobarbituric acid assay showed that ME resulted in a twofold increase of formation of malondialdehyde by liver microsomes incubated with FeCl(3) -ADP. ME produced a threefold increase of malondialdehyde formation by lung microsomes. The present study demonstrates that ME inhalation exposure differentially modulates cytochrome P-450 2B1 and antioxidant enzymes and increases susceptibility to lipid peroxidation in rat liver and lung.

Motorcycle exhaust particulates enhance vasoconstriction in organ culture of rat aortas and involve reactive oxygen species.

The effects of motorcycle exhaust particulate on vasoconstriction were determined using rat thoracic aortas under organ culture conditions treated with organic extracts of motorcycle exhaust particulate from a two-stroke engine. The motorcycle exhaust particulate extract (MEPE) induced a concentration-dependent enhancement of vasoconstriction elicited by phenylephrine in the organ cultures of both intact and endothelium-denuded aortas for 18 h. Nifedipine (an L-type Ca2+ channel blocker), manganese acetate (an inorganic Ca2+ channel blocker), and staurosporine (a nonselective protein kinase C inhibitor), but not the selective protein kinase C inhibitor chelerythrine, inhibited the enhancement of vasoconstriction by MEPE. Staurosporine has also been reported as a myosin light chain kinase (MLCK) inhibitor, so we tested whether the MLCK pathway was involved in the effect of MEPE. The results showed that ML-9 (a selective MLCK inhibitor) could inhibit the enhancement of vasoconstriction by MEPE. The phosphorylation of a 20-kDa myosin light chain in a primary culture of rat vascular smooth muscle cells was also enhanced by MEPE. Moreover, we also examined the role of reactive oxygen species (ROS) in the stimulatory effect of MEPE on vasoconstriction. The antioxidant N-acetylcysteine significantly inhibited the enhancement of vasoconstriction by MEPE. A time-dependent increase in ROS production by MEPE was also detected in primary cultures of vascular smooth muscle cells. These results indicate that MEPE induces a marked enhancement of vasoconstriction in aortas under organ culture conditions and imply that a ROS-Ca2+-MLCK pathway may be involved in this MEPE-induced response.