Peroxiredoxin 4 directly affects the male fertility outcome in porcine.

Peroxiredoxins (Prdxs) are known to play a critical role in regulating male fertility as antioxidant enzymes. Although several studies have suggested a close association between Prdxs and male fertility, few studies have explored the efficacy of Prdxs to predict male fertility. Therefore, the current study was designed to discover the most efficient biomarkers among the Prdxs with six isoforms. Our study showed a significant positive correlation between the litter size and the levels of PRDX 4 among all isoforms in spermatozoa. Subsequently, a regression analysis using a combination of markers was conducted to increase efficacy for fertility prediction. Nevertheless, PRDX4 had the highest efficacy compared to other combination models to predict litter size. The prediction accuracy of male fertility was further evaluated through receiver operating characteristic curve analysis, which showed that PRDX 4 could predict the litter size with high overall accuracy of 95%. Moreover, litter size was increased by 1.55 piglets after predicting high litter size using PRDX 4. This is the first study to comprehensively elucidate the role of all isoforms of PRDXs on male fertility to the best of our knowledge. PRDX 4 was tested and evaluated up to a practical level. Data here reported suggesting PRDX 4 marker allowed the highest accuracy for male fertility prediction and diagnosis, leading to a measurable improvement in the male fertility outcome.

Comparison of markers predicting litter size in different pig breeds.

To overcome the limitations of conventional analysis of male fertility in animals and humans, proteomic studies have been performed to develop fertility-related biomarkers for prognosis and diagnosis of male fertility. However, the studies were focused on specific species or breeds. Therefore, a study is required to validate whether fertility-related markers would apply to other breeds in pigs. In this study, previously developed fertility-related biomarkers from Landrace were validated to use for prognosis of male fertility in commercially available breeds. Expression level of eight biomarkers in non-capacitated and capacitated (C) spermatozoa from Yorkshire and Duroc boars was analyzed. And then, to explore the validity of these markers for prognosis of male fertility, i.e. litter size, artificial insemination was performed. Among them, RAB2A (NC) and UQCRC1 (NC) turned out to be highest efficient markers for Yorkshire. RAB2A (C) was most efficient marker for Duroc. Average litter size has increased as much as 1.41 live born after prediction using eight fertility-related biomarkers in Yorkshire. In addition, average 2.52 litter size was increased after prediction using eight fertility-related biomarkers in Duroc. Average litter sizes were especially highly increased after prediction of fertility using RAB2A (NC) in Yorkshire (1.57 piglets) and TPI (NC) in Duroc (3.14 piglets), respectively. As a result, all biomarkers were significantly correlated with litter size. However, overall accuracy to predict litter size in three breeds was different in response with each marker. Average litter size after artificial insemination was also significantly affected by marker selection. Therefore, this study suggests that developed fertility-related markers may be used for prognosis and diagnosis of male fertility irrespective of breed. However, selection of efficient markers for breeds should be considered to obtain more accurate and efficient outcomes.

Effect of lifestyles on the blood mercury level in Korean adults.

Mercury (Hg) is widely distributed in the environment and oral exposure is a main route in the general population. In this study, we estimated the dietary intake of Hg and its relationship with blood Hg levels in Korean adults. The study subjects were recruited from three different districts (rural: 189, coastal: 208 and urban: 184). We used a general questionnaire to collect information about demographic factors, lifestyles and diet. Dietary habits were studied using the 24-h recall method. The estimation of Hg intake was performed using the database of Hg contents in 128 Korean foods based on the previous studies. Blood Hg was analyzed using Direct Mercury Analyzer with the gold-amalgam method. Daily intake of Hg by diet was estimated at 13.57 µg (0.22 µg/kg body weight). The geometric mean Hg concentration in whole blood was 3.92 µg/L. Blood Hg level and Hg intake by diet was higher in coastal areas than in urban or rural areas, respectively. Blood Hg level correlated with the intake of Hg consumed from diet. Seafood was highly responsible and account for 75.6% of total dietary Hg intake. In this study, blood Hg concentrations were found to be significantly affected by sex, age, individual lifestyles and especially the amount of seafood intake, which might play an important role in determining blood Hg levels in Korean adults.

Management of gastric epithelial neoplasia in patients requiring esophagectomy for esophageal cancer.

Esophageal squamous cell carcinoma is occasionally associated with malignancies located in other regions of the alimentary tract, as well as in the head, neck, and upper respiratory tract. The stomach is most commonly used for reconstruction of the alimentary tract after esophagectomy for esophageal cancer. When synchronous tumors are located in the stomach, it is often unsuitable for use in esophageal reconstruction. In such cases, an invasive procedure involving anastomosis between the esophagus and the colon must be performed. However, this procedure is associated with a high incidence of mortality and morbidity. Seven patients with synchronous esophageal cancer and gastric epithelial neoplasia were encountered. First, endoscopic submucosal dissection (ESD) was performed for the gastric epithelial neoplasia. Then, following successful ESD, Ivor-Lewis esophagectomy for esophageal cancer was planned 1 to 2 weeks later. A total of 11 gastric epithelial lesions were found in seven patients. En bloc resection by ESD was possible in all 11 lesions and histologically complete resection was achieved in all 11 lesions. Follow-up endoscopy was done 1-2 weeks after ESD; six patients with well-healing ulcers underwent esophagectomy the next day (8 or 15 days after ESD). In one patient with a poorly healed ulcer, a second follow-up endoscopy was done 1 week later and then esophagectomy was performed the next day (22 days after ESD). Post-surgical complications related to ESD, such as bleeding or mediastinal leak, were not seen in any of the seven patients. In patients with synchronous esophageal cancer and gastric epithelial neoplasia, ESD for gastric epithelial neoplasia followed by Ivor-Lewis esophagectomy 1 to 2 weeks later is an effective choice of treatment.

Mechanically and structurally robust sulfonated block copolymer membranes for water purification applications.

The effective removal of ionic pollutants from contaminated water using negatively charged nanofiltration membranes is demonstrated. Block copolymers comprising polystyrene (PS) and partially hydrogenated polyisoprene (hPI) were synthesized by varying chain architectures. A one step procedure of cross-linking (hPI blocks) and sulfonation reactions (PS chains) was then carried out, which was revealed as an effective method to enhance mechanical integrity of membranes while hydrophilic sulfonated chains remain intact. In particular, the control of chain architecture allows us to create a synergetic effect on optimizing charge densities of the membrane, water permeability, and mechanical integrity under water purification conditions. The best performing membrane can almost completely (>99%) reject various divalent cations and also show NO(3)(-) rejection > 85% and Na(+) rejection > 87%. Well defined nanostructures (tens of nanometers) as well as the periodically arranged water domains (a few nanometers) within hydrophilic phases of the hydrated membranes were confirmed by in situ neutron scattering experiments.

Effects and neuro-toxic mechanisms of 2, 2', 4, 4', 5, 5'-hexachlorobiphenyl and endosulfan in neuronal stem cells.

Endocrine disrupters are exogenous compounds thought to mimic the action of estrogen or other hormones and influence endocrine activity in the body (Juberg, 2000). These chemicals have adverse effects not only in the reproductive system but also in the central nervous system during development and throughout life. Polychlorinated biphenyls (PCBs) are a class of environmentally persistent and widespread halogenated hydrocarbons. It has been reported that PCBs are potential neurotoxicants. Endosulfan is an organochlorine insecticide that is extensively used to control pests in vegetables, cotton, and fruits. To determine the effect of 2, 2', 4, 4', 5, 5',-hexachlorobiphenyl(2, 4, 5-HCB) and endosulfan on embryo nervous system, we isolated neural stem cells from rat brain at embryonic day 17. Isolated neural stem cells showed pluripotenty. Stem cells could differentiate into neurons and glia. Neurite formation in endosulfan and 2, 4, 5-HCB treated cells. And it appeared to be decreased as compared with that in untreated cells. In order to know the neuro-toxic mechanisms of 2, 4, 5-HCB and endosulfan in neuronal stem cells, we investigated mitogen-activated protein kinase activity (MAPK) and gap junctional intercellular communication (GJIC). Endosulfan decreased the MAPK activity in dose dependent manner. Endosulfan and 2, 4, 5-HCB inhibited GJIC compared to the untreated cell by scrape loading dye transfer (SL/DT). 2, 4, 5-HCB and endosulfan decreased the expression of connexin 43 in dose dependent manner. These results indicated that 2, 4, 5-HCB and endosulfan may inhibit differentiation and proliferation of neural stem cells and gap junctional intercellular communication which play a crucial role in the maintenance of cellular homeostasis.

Genomic imbalance in rat mammary gland carcinomas induced by 2-amino-1-methyl-6-phenylimidazo(4,5-b)pyridine.

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), a compound found in cooked meat, is a mammary gland carcinogen in female Sprague-Dawley rats. PhIP-induced rat mammary gland carcinomas were examined for mutations in several genes (exons) known to regulate cell growth and apoptosis, including p53 (4-8), p21(Waf1) (coding region), Apc (14, 15), B-catenin (3), E-cadherin (9,13,15), Bcl-x (coding region), Bax (3), IGFIIR (28), and TGFBIIR (3). DNA from 30 carcinomas was examined by single-strand conformation polymorphism analysis, but no mutations were detected in these genes or gene regions. DNA from carcinomas and matching normal tissue were further screened for allelic imbalance by using a polymerase chain reaction-based approach with primers to known microsatellite regions located throughout the rat genome. Of 53 markers examined, 12 revealed allelic imbalance. Microsatellite instability (MSI) was detected at two markers, one on chromosome 4 and one on chromosome 6. Sixty-five percent and 96% of all carcinomas examined (N=23) showed MSI at these loci on chromosomes 4 and 6, respectively, supporting the notion that MSI plays a role in PhIP-induced mammary carcinogenesis. Loss of heterozygosity (LOH), an indication of a possible tumor suppressor gene, was observed at 10 markers distributed on chromosomes 3, 10, 11, 14, and X. The frequency of LOH at these markers was 75-94%, supporting that the regions of allelic imbalance were largely similar for the PhIP-induced carcinomas examined in this study. When PhIP-induced carcinomas from rats placed on high-fat and low-fat diet were compared, no unique regions of allelic imbalance or statistical differences in the frequency of allelic imbalance were observed. Therefore, the high-fat diet, known to be a promoter of PhIP-induced rat mammary carcinogenesis, did not appear to influence allelic imbalance in the carcinomas. Interestingly, 7,12-dimethylbenz[a]-anthracene-induced mammary carcinomas did not show allelic imbalance at 11 of the 12 loci that showed allelic imbalance in PhIP-induced carcinomas. These findings suggest that distinct chemical carcinogens induce different patterns of allelic imbalance during rat mammary carcinogenesis. Since several of the known genes involved in carcinogenesis did not harbor mutations in PhIP-induced carcinomas, further studies are needed to clarify the critical genes involved in PhIP-induced mammary carcinogenesis and to determine whether regions of LOH harbor potentially novel tumor suppressor genes involved in this disease.

Immature uterotrophic assay is more sensitive than ovariectomized uterotrophic assay for the detection of estrogenicity of p-nonylphenol in Sprague-Dawley rats.

Many efforts have been made to develop assays for detecting endocrine disrupters (EDs). Among them, uterotrophic assay has been known efficient for detecting EDs, especially estrogenic compounds. This study was performed to compare the immature uterotrophic assay with an ovariectomized assay using p-nonylphenol (NP), a weakly estrogenic compound. NP was given to either immature or ovariectomized rats subcutaneously or orally (only immature) at doses of 10, 100, and 1000 mg/kg for 3 days. After treatment with NP, the rats were examined for parameters such as uterine weight, uterine weight per body weight ratio, luminal epithelial height of uterus and vagina, diameter of uterine ducts, and number of uterine glands. Both systems were shown to increase uterine weight in a dose-dependent manner. In the immature system (subcutaneous injection), uterine weight, diameter of uterine duct and vaginal luminal epithelial height were significantly increased at 100 mg/kg/day, while in the ovariectomized system these parameters were not significant at the same dose (except for vaginal luminal epithelial height). These results suggest that the immature system (subcutaneous injection) might be most sensitive to detecting a weakly estrogenic compound and that the measurement of vaginal epithelium is a good end-point.

Carcinogenicity and mutagenicity of heterocyclic amines in transgenic mouse models.

Double transgenic mice bearing fusion genes consisting of mouse albumin enhancer/promoter-mouse c-myc cDNA and mouse metallothionein1 promoter-human TGFalpha cDNA were generated to investigate the interaction of these genes in hepatic oncogenesis and to provide a general paradigm for characterizing both the interaction of nuclear oncogenes and growth factors in tumorigenesis. In addition, these mice provide an experimental model to test how environmental chemicals might interact with the c-myc and TGFalpha transgenes during the neoplastic process. Treatment of the double transgenic mice with both genotoxic agents such as diethylnitrosamine and 2-amino-3-methylimidazo-[4,5-f]quinoline (IQ) as well as the tumor promoter phenobarbital greatly accelerated the neoplastic process. To investigate the role of mutagenesis in the carcinogenic process, 2-amino-3,8-dimethyl-imidazo[4,5-f]quinoxaline (MeIQx) induced mutagenesis and hepatocarcinogenicity was examined in C57BL/lacZ (Muta Mice) and double transgenic c-myc/lacZ mice that carry the lacZ mutation reporter gene. The MelQx hepatocarcinogenicity was associated with an increase in in vivo mutagenicity as scored by mutations in the lacZ reporter gene. These results suggest that transgenic mouse models may provide important tools for testing both the carcinogenic potential of environmental chemicals and the interaction/cooperation of these compounds with specific genes during the neoplastic process.

Constitutive expression and induction of CYP1B1 mRNA in the mouse.

Previous studies appeared to indicate that CYP1B1 was not constitutively expressed in mouse liver. In our laboratory, we demonstrated using aromatic hydrocarbon-responsive receptor knock-out (AHR-(-)/-) mice that both piperonyl butoxide (PBO) and acenaphtyhlene (ACN) are AHR-independent inducers of murine CYP1A2 and CYP1B1 mRNA. In the current study, we demonstrate both constitutive levels and induction of CYP1B1 in mouse liver. The induction of CYP1B1 mRNA by PBO or ACN was higher in DBA/2 (Ahrd) than in C57BL/6 (Ahrb-1) mice, while 3-methylcholanthrene induced CYP1B1 more in C57BL/6 than in DBA/2 mice. These results suggest that CYP1B1 may also be induced by more than one mechanism. In addition, constitutive expression of CYP1B1 was detected in liver, kidney, and lung of untreated C57BL/6 mice. There was no gender difference in CYP1B1 expression; however, in C57BL/6 mice, the kidney contained less CYP1B1 than either liver or lung.

In vivo mutagenicity and hepatocarcinogenicity of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) in bitransgenic c-myc/lambda lacZ mice.

2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) is a mutagenic and carcinogenic heterocyclic amine found in cooked meat. Hepatic DNA adduct formation, in vivo mutagenicity, and hepatocarcinogenicity of MeIQx were examined in mice harboring the lacZ mutation reporter gene (Muta mice) and bitransgenic mice overexpressing the c-myc oncogene. C57Bl/lambda lacZ and bitransgenic c-myc (albumin promoter)/lambda lacZ mice were bred and weaned onto an American Institute of Nutrition-76-based diet containing 0.06% (w/w) MeIQx or onto control diet. After 30 weeks on diet, only male bitransgenic mice on MeIQx developed hepatocellular carcinoma (100% incidence). By 40 weeks, hepatic tumor incidence was 100%/75% (17%/0%) and 44%/17% (0%/0%) in male c-myc/lambda lacZ and C57Bl/lambda lacZ mice who were given MeIQx (or control) diet, respectively, supporting a synergism between MeIQx and c-myc overexpression in hepatocarcinogenesis. At either time point, mutant frequency in the lacZ gene was at least 40-fold higher in MeIQx-treated mice than in control mice of either strain. These findings suggest that MeIQx-induced hepatocarcinogenesis is associated with MeIQx-induced mutations. Elevated mutant frequency in MeIQx-treated mice also occurred concomitant with the formation of MeIQx-guanine adducts, as detected by the 32P-postlabeling assay. Irrespective of strain or diet, sequence analysis of the lacZ mutants from male mouse liver showed that the principal sequence alterations were base substitutions at guanine bases. Adenine mutations, however, were detected only in animals on control diet. MeIQx-fed mice harboring the c-myc oncogene showed a 1.4-2.6-fold higher mutant frequency in the lacZ gene than mice not carrying the transgene. Although there was a trend toward higher adduct levels in c-myc mice, MeIQx-DNA adduct levels were not significantly different between c-myc/lambda lacZ and C57Bl/lambda lacZ mice after 30 weeks on diet. Thus, it seemed that factors in addition to MeIQx-DNA adduct levels, such as the enhanced rate of proliferation associated with c-myc overexpression, may have accounted for a higher mutant frequency in c-myc mice. In the control diet groups, the lacZ mutant frequency was significantly higher in c-myc/lambda lacZ mice than in C57Bl/lambda lacZ mice. The findings are consistent with the notion that c-myc overexpression is associated with an increase in mutagenesis. The mechanism for the synergistic effects of c-myc overexpression on MeIQx hepatocarcinogenicity seems to involve an enhanced expression of MeIQx-induced mutations.

Regulation of mouse liver flavin-containing monooxygenases 1 and 3 by sex steroids.

Based on enzyme activity, protein levels, and mRNA levels, we have previously demonstrated the female-predominant, female-specific, and gender-independent expression in mouse liver of FMO forms 1, 3, and 5, respectively. This study investigated the roles of testosterone, 17 beta-estradiol, and progesterone in the regulation of hepatic FMOs. FMO expression was examined in gonadectomized CD-1 mice, normal CD-1 mice receiving hormonal implants, and gonadectomized mice receiving various hormonal treatments. Following castration of males, hepatic FMO activity levels were significantly increased and serum testosterone levels significantly decreased; however, administration of physiological levels of testosterone to castrated animals returned FMO activity and testosterone concentrations to control levels. When sexually intact and ovariectomized female mice were treated with testosterone, their hepatic FMO activity levels were reduced to those of their male counterparts, concomitant with high serum testosterone levels. In males, castration dramatically increased FMO3 and FMO1 expression, and testosterone replacement to castrated males resulted in ablation of FMO3 expression. In addition, testosterone administration to females (sexually intact and gonadectomized animals) reduced FMO1 expression and obviated FMO3 expression. In females, ovariectomy alone slightly reduced FMO activity, indicative of a possible stimulatory role of female sex steroids; however, female FMO isozyme expression was relatively unchanged, and hormone replacement therapy to ovariectomized females had no discernible effect. In males and females, FMO5 levels were unaffected by gonadectomy or hormone administration, thus indicating a sex hormone-independent mechanism of regulation for this isoform. Interestingly, FMO1 protein levels were increased in sexually intact males following treatment with 17 beta-estradiol; however, only a slight increase in FMO3 protein level was observed. No positive hormone effectors of female FMO expression were identified.

Regulation of hepatic CYP1A isozymes by piperonyl butoxide and acenaphthylene in the mouse.

The regulation of CYP1A1 and CYP1A2 isozymes by piperonyl butoxide (PBO) and acenaphthylene (ACN) was studied in the liver of male C57BL/6 and DBA/2 mice. These two cytochrome P450 genes are known to be regulated by the aromatic hydrocarbon-responsive receptor (AHR); however, it has been suggested that CYP1A2 is also induced by an AHR-independent mechanism. In this study, PBO induced hepatic CYP1A1 considerably more in C57BL/6 (Ahrb-I) than in DBA/2 (Ahrd) mice. In addition, the superinduction of CYP1A1 in wildtype hepa1c1c7 cells, which is AHR-dependent, resulted from PBO and cycloheximide treatment of the cells. In other studies in this laboratory using AHR knock-out (AHR-/-) mice, a hybrid of 129/SV and C57BL/6 strains, no induction of CYP1A1 occurred with PBO or ACN. [D.-Y. Ryu, P.E. Levi, P. Fernandez-Salguero, F.J. Gonzalez, E. Hodgson, Mol. Pharmacol., 50 (1996) 443-446.] ACN, however, did not induce CYP1A1 under the experimental conditions used. These results suggest that PBO, but not ACN, induces CYP1A1 through a weak activation of AHR. On the other hand, hepatic CYP1A2 mRNA and hnRNA were induced by PBO in both C57BL/6 and DBA/2 strains, but were not induced by ACN, a strong inducer of CYP1A2 in the B6C3F1 strain. However, both PBO and ACN induced CYP1A2 in AHR-/- mice. It is assumed, therefore, that the transcriptional induction of CYP1A2 by PBO and ACN is AHR-independent. In addition, the induction of CYP1A2 by ACN depends upon the strain of mice. Immunohistochemical studies for CYP1A1/CYP1A2 apoproteins showed that PBO induced CYP1A1/CYP1A2 around the central veins as did 3-methylcholanthrene (3-MC). The induction of CYP1A1/CYP1A2 by ACN, however, was not observed, consistent with the northern blot results.

Piperonyl butoxide and acenaphthylene induce cytochrome P450 1A2 and 1B1 mRNA in aromatic hydrocarbon-responsive receptor knock-out mouse liver.

It has been suggested that acenaphthylene (ACN), piperonyl butoxide (PBO) and other methylenedioxyphenyl (benzodioxole) compounds can function as aromatic hydrocarbon-responsive receptor (AHR)-independent inducers of the cytochrome P450 (CYP) 1A2 in mouse liver. Although much indirect evidence has supported this hypothesis, direct proof was lacking until the present study. PBO and ACN were used to examine the expression of CYP1A1, CYP1A2 and CYP1B1 in mouse liver. These three CYP isozymes are included in the AHR battery of proteins. In this study, AHR knock-out mice were dosed intraperitoneally with PBO (200 mg/kg) or ACN (100 mg/kg). Induction of hepatic CYP1A1 by PBO or ACN was not detected by northern blots. In contrast, both CYP1A2 and CYP1B1 mRNA, constitutively expressed at low levels in this tissue, were induced by each compound in the livers of AHR knock-out mice. In addition, the use of heterogenous nuclear RNA reverse transcription-polymerase chain reaction procedures revealed that the transcriptional activities of CYP1A2 were increased by PBO and ACN treatments. These results show that AHR-independent pathway(s) can be involved in induction of CYP1A2 and CYP1B1.

Pesticide-metabolizing enzymes.

Pesticides are known to function as substrates, inhibitors and inducers of drug-metabolizing enzymes, with the same compound frequently acting in more than one of these roles. Current studies of phase I metabolism of pesticides include cytochrome P450 (P450) and the flavin-containing monooxygenase (FMO), with particular reference to individual isozymes. In mouse liver, the level of FMO1 is gender dependent, FMO3 is gender specific, while FMO5 appears to be gender independent. The isozyme specificity of methylenedioxyphenyl synergists for induction of P450 in mouse liver involves P450s 1A1, 1A2 and 2B10, including a non-Ah receptor-dependent mechanism for 1A2 induction. The substrate specificity of mouse and human P450 and FMO isozymes is discussed.

Biphasic responses in synergistic interactions.

Synergistic reactions, as exemplified by the methylenedioxyphenyl (benzodioxole) insecticide synergists, are one of the more studied interactions between toxicants. This group of chemicals includes, in addition to synergists such as piperonyl butoxide, carcinogens such as safrole and isosafrole and many compounds occurring naturally in foods, such as myristicin and piperine. These compounds may function as cytochrome P450 substrates, inhibitors and/or inducers. The biphasic curve in cytochrome P450 activities following a single dose is the result of an initial inhibitory phase followed by a phase of induction, with an eventual return to baseline levels. Both inhibition and induction may be isozyme-specific and different isozymes may be involved in the two activities of the same chemical. Details of these activities are presented and their significance is discussed.

Regulation of cytochrome P-450 isozymes CYP1A1, CYP1A2 and CYP2B10 by three benzodioxole compounds.

Three benzodioxole (BD) compounds were used to investigate the structural requirement for regulation of the cytochrome P450 isozymes, CYP1A1, CYP1A2 and CYP2B10, in mouse liver. Male mice (C57BL/6) were treated intraperitoneally for 3 days with 5-t-butyl-1,3-benzodioxole (t-BBD), 5-n-butyl-1,3-benzodioxole (n-BBD) and 5-(3-oxobutyl)-1,3-benzodioxole (o-BBD). t-BBD-induced liver microsomes showed the highest pentoxyresorufin O-dealkylation (PROD) activity, while o-BBD induced microsomes showed slightly higher activity in ethoxyresorufin O-deethylation (EROD), benzo[a]pyrene hydroxylation (BaP-OH) and acetanilide hydroxylation (Acet-OH) assays. In vitro enzyme inhibition assays showed that n-BBD inhibited EROD and Acet-OH activities more than either o-BBD or t-BBD, while PROD activity was evenly inhibited by all three compounds. Western and northern blots showed that CYP1A1 was not detectably induced by any of the three BD compounds. The levels of CYP1A2 protein and mRNA were increased in all three treated livers. In addition to CYP1A2 induction, t-BBD also induced the protein and mRNA for CYP2B10.