Michael J. Kohan

Effects of benzo[a]pyrene exposure on a fish population resistant to the toxic effects of dioxin-like compounds ☆

Effects of a model polycyclic aromatic hydrocarbon (PAH) were compared in populations of the estuarine fish Fundulus heteroclitus indigenous to a reference site and one highly contaminated with polychlorinated biphenyls (PCBs) and other compounds. The fish population resident to the PCB-contaminated site is genetically resistant to those PCB congeners categorized as dioxin-like compounds (DLCs) that act through the aryl hydrocarbon receptor (AHR). In response to DLC exposures, these DLC-resistant fish showed poor inducibility for enzymes known to be regulated by the AHR pathway and important for the metabolism of xenobiotics including some PAHs that also act as AHR agonists. Therefore, a laboratory study using the model PAH, benzo[a]pyrene (BaP), was conducted to evaluate how PAHs might affect these wild fish populations that differed in their inherent sensitivities to DLCs and in their tissue concentrations of contaminants. Following BaP treatment, the activities of two xenobiotic metabolizing enzymes and the concentrations of BaP-DNA adducts, as measured using the 32P-postlabeling method, were lower in the livers of DLC-resistant than reference fish. These results suggest that DLC-resistance could provide protection following chronic exposures to PAHs from the long-term consequences of DNA adduct formation, such as cancer. Alternatively, reduced metabolism and elimination of toxic or photo-activated PAHs could have acute consequences to the health and reproduction of these DLC-resistant fish and their progeny. These fish populations provide useful models to evaluate the potential costs and benefits of genetic adaptation in wildlife populations subject to anthropogenic stress.

A potential microRNA signature for tumorigenic conazoles in mouse liver.

Triadimefon, propiconazole, and myclobutanil are conazoles, an important class of agricultural fungicides. Triadimefon and propiconazole are mouse liver tumorigens, while myclobutanil is not. As part of a coordinated study to understand the molecular determinants of conazole tumorigenicity, we analyzed the microRNA expression levels in control and conazole‐treated mice after 90 d of administration in feed. MicroRNAs (miRNAs) are small noncoding RNAs composed of approximately 19–24 nucleotides in length, and have been shown to interact with mRNA (usually 3′ UTR) to suppress its expression. MicroRNAs play a key role in diverse biological processes, including development, cell proliferation, differentiation, and apoptosis. Groups of mice were fed either control diet or diet containing 1800 ppm triadimefon, 2500 ppm propiconazole, or 2000 ppm myclobutanil. MicroRNA was isolated from livers and analyzed using Superarray whole mouse genome miRNA PCR arrays from SABioscience. Data were analyzed using the significance analysis of microarrays (SAM) procedure. We identified those miRNAs whose expression was either increased or decreased relative to untreated controls with q ≤ 0.01. The tumorigenic conazoles induced many more changes in miRNA expression than the nontumorigenic conazole. A group of 19 miRNAs was identified whose expression was significantly altered in both triadimefon‐ and propiconazole‐treated animals but not in myclobutanil‐treated animals. All but one of the altered miRNAs were downregulated compared to controls. This pattern of altered miRNA expression may represent a signature for tumorigenic conazole exposure in mouse liver after 90 d of treatment. Published 2010 Wiley‐Liss, Inc.

Polychlorinated biphenyl‐degrading pseudomonads: Survival in mouse intestines and competition with normal flora

Although naturally occurring and mutant organisms, historically, have been released into the environment for various purposes, health concerns associated with the release of microorganisms have recently resurfaced. Federal agencies have been given the task of reassuring society that any released organisms are not likely to produce adverse health effects. Methods, therefore, for evaluating the potential health effects due to environmental release of mutant and genetically engineered microorganisms are under investigation. A mouse model was developed that examines morbidity, mortality, and more indirect effects such as colonization potential of the intestinal tract, as well as competition with and alteration of the intestinal microbiota populations. The Pseudomonas spp. used in this study were isolated from a commercial product and used for degrading polychlorinated biphenyls. Mice were dosed individually with 10(3), 10(6), and 10(9) colony-forming units of each microorganism. At specific time intervals the intestines were removed and examined for the presence of the dosed microorganism. At the two higher doses, 10(6) and 10(9) colony-forming units, P. maltophilia strain BC6 and two P. aeruginosa strains, BC16 and BC18, were recoverable 48 h after dosing. The naturally occurring P. aeruginosa strain, PAMG, isolated from a mouse intestinal homogenate produced a similar response. Statistical analysis indicated that in some of the dosed animals, an alteration in the distribution of normal intestinal microflora occurred. Pseudomonas maltophilia strain BC6 and P. aeruginosa strains BC16 and BC17 caused a change in the obligately anaerobic predominantly gram-negative rod counts, and P. aeruginosa strain BC17 produced a dose effect on the total anaerobic count at the 10% confidence level. The total aerobic count was unaffected by the presence of the dosed pseudomonads.

Oral treatment of Fischer 344 rats with weathered crude oil and a dispersant influences intestinal metabolism and microbiota.

When oil is spilled into aquatic systems, chemical dispersants frequently are applied to enhance emulsification and biological availability. In this study, a mammalian model system was used to determine the effect of Bonnie Light Nigerian crude oil, weathered for 2 d with continuous spraying and recirculation, and a widely used dispersant, Corexit (Cx) 9527, on intestinal microbial metabolism and associated populations. To determine the subchronic dose, concentrated or diluted (1:2, 1:5, 1:10, 1:20) Cx9527 or oil was administered by gavage to Fischer 344 rats and the effect on body weight was determined. Next, rats were treated for 5 wk with oil, dispersant, or dispersant + oil. Body and tissue weights, urine mutagenicity, and the impact on the intestinal microflora and three microbial intestinal enzymes linked to bioactivation were determined in the small and large intestines and cecum. Two tested dispersants, Cx9527 and Cx9500, were toxic in vitro (1:1,000 dilution), and oil was not mutagenic in strains TA98 and TA100(+/-S9). None of the treated rats produced urine mutagens detected by TA98 or TA100. Undiluted dispersant was lethal to rats, and weight changes were observed depending on the dilution, whereas oil generally was not toxic. In the 5-wk study, body and tissue weights were unaffected at the doses administered. Small-intestinal levels of azoreductase (AR), beta-glucuronidase (BG), and nitroreductase (NR) were considerably lower than cecal and large-intestinal activities at the same time point. A temporal increase in AR activity was observed in control animals in the 3 tissues examined, and large-intestinal BG activity was elevated in 3-wk controls. No significant changes in cecal BG activity were observed. Oil- or dispersant-treated rats had mixed results with reduced activity at 3 wk and elevated activity at 5 wk compared to controls. However, when the dispersant was combined with oil at 3 wk, a reduction in activity was observed that was similar to that of dispersant alone. One-week nitroreductase activity in the small intestine and cecum was unaffected in the three treatment groups, but elevated activity was observed in the large intestines of animals treated with oil or dispersant. The effect of the combination dose was not significantly different from the control value. Due to experimental error, no 3- or 5-wk NR data were available. By 5 wk of treatment, enterobacteria and enterococci were eliminated from ceca of oil-treated rats. When oil was administered in combination with dispersant, an apparent protective effect was observed on the enterococci and lactose-fermenting and nonfermenting enterobacteria. A more detailed analysis at the species level revealed qualitative differences dependent on the treatment. This study suggests that prolonged exposure of mammals to oil, dispersant, or in combination impacts intestinal metabolism, which ultimately could lead to altered detoxification of oil constituents and coexposed toxicants.

Potentiation of 2,6-dinitrotoluene genotoxicity in fischer 344 rats by pretreatment with pentachlorophenol

Abstract The organochlorine pesticide, pentachlorophenol, a potent sulfotransferase inhibitor, reportedly reduces the binding of 2,6-dinitrotoluene, an industrial hepatocarcinogen to hepatic DNA by 95% after a single i.p. injection. Activation of 2,6-dinitrotoluene to genotoxic metabolites involves enzymes in both the liver and the intestinal flora. Since pentachlorophenol also has bactericidal activity and induces hepatic mixed function oxidase activity after longer treatment, the effect of pentachlorophenol on intestinal enzyme activity and the biotransformation of 2,6-dinitrotoluene to genotoxic metabolites was studied after 1, 2, 4, and 5 weeks of treatment. Male Fischer 344 rats were dosed daily, by gavage, with either 20 mg/kg pentachlorophenol or the peanut oil vehicle. After 1, 2, 4, and 5 weeks, select control and treated animals were injected p.o. with 75 mg/kg 2,6-dinitrotoluene and transferred to metabolism cages, where urine was collected for 24 hr and tested for mutagenic activity by the Ames Salmonella typhimurium reversion assay. At 2 and 4 weeks, six control and six treated animals were sacrificed and nitroreductase, azo reductase, β-glucuronidase, dechlorinase, and dehydrochlorinase activities were analyzed in homogenates of the small intestine, large intestine, and cecum. At 5 weeks, hepatic DNA adduct formation was assayed by the 32 P-postlabeling of DNA. Results from this study indicated that pentachlorophenol accelerated the biotransformation of 2,6-dinitrotoluene to genotoxic metabolites and potentiated the formation of 2,6-dinitrotoluene-induced DNA adducts in the liver. This is the first report of a chemical interaction leading to increased DNA adduct formation and indicates that chemical interactions could be important to risk assessment since they alter the relationship between exposure, dose, and the effect of genotoxicants.

COLONIZATION AND CLEARANCE OF ENVIRONMENTAL MICROBIAL AGENTS UPON INTRANASAL EXPOSURE OF STRAIN C3H/HeJ MICE

Environmental dissemination of biotechnology agents is becoming a common practice. Most applications use historically innocuous species; however, potential health effects of individual products are not scrutinized unless they contain genetically engineered microorganisms. In order to investigate possible health concerns, four surrogate microbial agents were studied in vivo. Male C3H/HeJ (endotoxin-resistant) mice were administered intranasally (i.n.) with approximately 10(7) Pseudomonas aureofaciens, Burkholderia cepacia, P. fluorescens, or P. putida. To determine clearance of the dosed bacterial strains, lungs, small intestine, large intestine, cecum, mesenteric lymph nodes (MLN), spleen, and liver were homogenized individually, plated, and dilutions inoculated onto selective media. Pseudomonas fluorescens and P. putida were eliminated from the lungs by 2 d posttreatment, and P. aureofaciens was not detected in the lungs by 5 d posttreatment. Burkholderia cepacia was reisolated from the lungs and cecum for the experimental duration (14 d). Translocation to extraintestinal sites (MLN, spleen, and liver) also occurred. Burkholderia cepacia was recovered from the MLN for 10 d after treatment of mice. Pulmonary exposure to several bacterial strains resulted in unexpected mortality. Pseudomonas aureofaciens was lethal at the lowest dose (8.26 x 10(6) CFU/ mouse), while P. fluorescens and B. cepacia were fatal at higher doses (6.15 x 10(8) CFU/mouse and 1.34 x 10(8) CFU/mouse, respectively). By using the model described in this study, human safety issues can be more easily addressed and evaluated.

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes†

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord‐region dihydrodiol, trans‐DB[a,l]P‐11,12‐diol, to syn‐ and anti‐DB[a,l]P‐11,12‐diol‐13,14‐epoxides (syn‐ and anti‐DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin‐layer chromatography, and high‐pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE‐DNA adducts and unidentified polar adducts at rates threefold and 17‐fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti‐ and syn‐DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti‐DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn‐DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley‐Liss, Inc.

Potentiation of 2,6‐dinitrotoluene genotoxicity in fischer 344 rats by pretreatment with coal tar creosote

Pretreatment of male Fischer 344 rats for 5 wk with coal tar creosote, a coal distillation product that is widely used as a wood preservative, potentiated the excretion of urinary mutagens in 2,6-dinitrotoluene (DNT) treated rats. Creosote increased the bioactivation of DNT to significantly greater levels of urinary genotoxic metabolites and/or formed DNA adducts in the liver. A significant increase in the excretion of mutagenic DNT metabolites was observed after the first week of creosote treatment, peaked at wk 3, and then decreased by 33% after 5 wk of treatment. Nevertheless, there was a significant increase (66%) in the formation of DNT-derived DNA adducts in the livers of rats treated with DNT plus creosote at wk 5. Increased cecal beta-glucuronidase activity and reduced small intestinal nitroreductase activity may play roles in the bioactivation of DNT. The excretion of mutagenic DNT metabolites supplies useful information about the bioactivation of DNT; it does not provide a useful index of DNT-derived hepatic DNA adduct formation. Such interactions could be important to predictive risk assessment because the overall cancer risk of such chemical mixtures may exceed the sum of the component risks.

Metabolism of 1‐nitropyrene by human, rat, and mouse intestinal flora: Mutagenicity of isolated metabolites by direct analysis of HPLC fractions with a microsuspension reverse mutation assay

The metabolism of [14C]-1-nitropyrene by human, rat and mouse intestinal microflora and a bioassay-directed chemical analysis of the isolated metabolites by assaying HPLC fractions with a microsuspension reverse mutation assay were examined. [14C]-1-Nitropyrene was metabolized by human, rat, and mouse intestinal microflora to 1-aminopyrene, N-acetyl-1-aminopyrene, N-formyl-1-aminopyrene, and two unknown metabolites identified as A and B. The predominant metabolite produced by human, rat, or mouse intestinal microflora following a 12-h incubation with [14C]-1-nitropyrene was 1-aminopyrene, which accounted for 93, 79, and 88% of the total 14C, respectively. Only minor amounts of N-formyl-1-aminopyrene (1.4, 1.2, and 1.0%), N-acetyl-1-aminopyrene (4.4, 3.0, and 3.4%), unknown A (1.0, 1.2, and 1.0%), and unknown B (3.3, 5.0, and 1.2%) were detected. These data suggest that a similar mechanism exists in the biotransformation of 1-nitropyrene by intestinal microflora from all three sources. Direct mutagenicity analysis of the HPLC fractions produced by intestinal microflora with the microsuspension reverse mutation assay indicated that mutagenic fractions can be resolved using this methodology.

Mutagenicity of HPLC-fractionated urinary metabolites from 2,4,6-trinitrotoluene-treated Fischer 344 rats

The production and storage of explosives has resulted in the environmental accumulation of the mutagen 2,4,6‐trinitrotoluene (TNT). In order to characterize the production of mutagenic urinary metabolites, 6‐week old male Fischer 344 rats were administered 75 mg of TNT/kg or DMSO vehicle by gavage. The animals were placed into metabolism cages, and urine was collected for 24 hr. Following filtration, metabolites in the urine were deconjugated with sulfatase and β‐glucuronidase and concentrated by solid phase extraction. The eluate was fractionated by reverse‐phase high‐performance liquid chromatography (HPLC) using acetonitrile/water, and the fractions were solvent exchanged in DMSO by nitrogen evaporation. Each HPLC fraction was bioassayed in strains TA98, TA98NR, TA100, and TA100NR without metabolic activation using a microsuspension modification of the Salmonella histidine reversion assay. Fractions 3, 5–18, 21, 22, and 24–26 contained mutagens detected by strain TA98. In the nitroreductase‐deficient strain TA98NR, some mutagenic activity was lost; however, fractions 3, 6, 9–11, 15, and 25 clearly contained direct‐acting mutagens. Fewer fractions were positive in strain TA100 (9–16, 19, 20, and 25) with less activity observed in the nitroreductase deficient strain TA100NR (fractions 3, 12, 14, 15, and 25). Although some mutagenic activity coeluted with known TNT metabolite standards, there were still many unidentified mutagenic peaks. Environ. Mol. Mutagen. 30:298–302, 1997.