Administration of supplemental oxygen is frequently encountered in infants suffering from pulmonary insufficiency and in adults with acute respiratory distress syndrome. However, hyperoxia causes acute lung damage in experimental animals. In the present study, we investigated the roles of the Ah receptor (AHR) in the modulation of cytochrome P4501A (CYP1A) enzymes and in the development of lung injury by hyperoxia. Adult male wild-type [AHR (ϩ/ϩ)] mice and AHR-deficient animals [AHR (Ϫ/Ϫ)] were maintained in room air or exposed to hyperoxia (Ͼ95% oxygen) for 24 to 72 h, and pulmonary and hepatic expression of CYP1A and lung injury were studied. Hyperoxia caused significant increases in pulmonary and hepatic CYP1A1 activities (ethoxyresorufin O-deethylase) and mRNA levels in wild-type (C57BL/6J) AHR (ϩ/ϩ), but not AHR (Ϫ/Ϫ) mice, suggesting that AHR-dependent mechanisms contributed to CYP1A1 induction. On the other hand, hyperoxia augmented hepatic CYP1A2 expression in both wild-type and AHR (Ϫ/Ϫ) animals, suggesting that AHR-independent mechanisms contributed to the CYP1A2 regulation by hyperoxia. AHR (Ϫ/Ϫ) mice exposed to hyperoxia were more susceptible than wildtype mice to lung injury and inflammation, as indicated by significantly higher lung weight/body weight ratios, increased pulmonary edema, and enhanced neutrophil recruitment into the lungs. In conclusion, our results support the hypothesis that the hyperoxia induces CYP1A1, but not CYP1A2, expression in vivo by AHR-dependent mechanisms, a phenomenon that may mechanistically contribute to the beneficial effects of the AHR in hyperoxic lung injury.

Increases in abundance of cathepsin B transcript and protein correlate with increases in tumor grade and alterations in subcellular localization and activity of cathepsin B. The enzyme is able to degrade the components of the extracellular matrix (ECM) and activate other proteases capable of degrading ECM. To investigate the role played by this protease in the invasion of brain tumor cells, we transfected SNB19 human glioblastoma cells with a plasmid containing cathepsin B cDNA in antisense orientation. Control cells were transfected with vector alone. Clones expressing antisense cathepsin B cDNA exhibited signi®cant reductions in cathepsin B mRNA, enzyme activity and protein compared to controls. Matrigel Invasion assay showed that the antisense-transfected cells had a markedly diminished invasiveness compared with controls. When tumor spheroids containing antisense transfected SNB19 cells expressing reduced cathepsin B were co-cultured with fetal rat brain aggregates, invasion of fetal rat brain aggregates was signi®cantly reduced. Green Fluorescent Protein (GFP) expressing parental cells and antisense transfectants were generated for detection in mouse brain tissue without any postchemical treatment. Intracerebral injection of SNB19 stable antisense transfectants resulted in reduced tumor formation in nude mice. These results strongly support a role for cathepsin B in the invasiveness of human glioblastoma cells and suggest cathepsin B antisense may prove useful in cancer therapy. Oncogene (2001) 20, 3665 ± 3673.

The cytochrome P4501A enzymes play important roles in carcinogen metabolism. We reported previously that 3-methylcholanthrene (MC) elicits a persistent induction of hepatic, pulmonary, and mammary microsomal cytochrome P450 (P450) 1A enzymes for several weeks after MC withdrawal. In this study, we tested the hypothesis that CYP1A2, a liver-specific P450 isozyme, plays an important role in the mechanisms governing persistent CYP1A1 induction by MC in liver but not in extra-hepatic tissues such as lung, which is devoid of endogenous CYP1A2. Administration of wild-type (WT) or CYP1A2-null mice with MC (100 mol/kg i.p.) once daily for 4 days caused significant increases in hepatic CYP1A1/1A2 activities, apoprotein contents, and mRNA levels 1 day after carcinogen withdrawal compared with vehicle-treated controls. The induction persisted in the WT, but not CYP1A2-null animals, for up to 15 days. In the lung, MC caused persistent CYP1A1 induction for 15 days in both the genotypes. Since MC is almost completely eliminated by day 15, we hypothesize that CYP1A2 contributes to the up-regulation of CYP1A1 in liver, but not lung, by a novel mechanism, presumably involving a CYP1A2-dependent persistent metabolite. The studies demonstrate tissue-specific differences in the regulation of CYP1A by MC, a phenomenon that may have implications for human carcinogenesis caused by environmental chemicals.

There is significant human exposure to polycyclic aromatic hydrocarbons (PAHs), many of which are potent carcinogens in laboratory animals and are suspected human carcinogens. The PAHs are bioactivated by cytochrome P450 (CYP)1A1/ 1B1 enzymes to reactive intermediates that bind to DNA, a critical step in the initiation of carcinogenesis. The Ah receptor (AHR) plays a critical role in the induction of CYP1 enzymes (i.e., CYP1A1, 1A2 and 1B1) by PAHs such as benzo[a]pyrene (BP) and 3-methylcholanthrene (MC). In our investigation, we tested the hypothesis that AHR-null animals are less susceptible to PAH-induced DNA adduct formation than wild-type animals. Wild-type [AHR (؉/؉)] mice or mice lacking the gene for the AHR were treated with a single dose (100 mol/kg) of BP or MC, and hepatic DNA adducts were analyzed by 32 P-postlabeling. BP induced multiple hepatic DNA adducts in wild-type as well as AHR-null animals, suggesting the existence of AHR-independent mechanisms for BP metabolic activation. On the other hand, DNA adduct formation was markedly suppressed in AHRnull animals exposed to MC, although the major MC-DNA adduct was produced in these animals. Hepatic activities and apoprotein contents of 7-ethoxyresorufin O-deethylase (EROD) (CYP1A1) and 7-methoxyresorufin O-demethylase (MROD) (CYP1A2) activities were markedly induced by BP and MC in the wild-type, but not, in AHR-null animals. CYP1B1 expression was also induced, albeit to a lesser extent by the PAH MC, but not BP, in the wild-type animals. In conclusion, these results demonstrate the existence of AHRand CYP1A1-independent mechanisms of PAH metabolic activation in mouse liver, a phenomenon that may have important implications for PAH-mediated carcinogenesis. Human cancer is thought to arise from interplay of endogenous factors and environmental exposures. 1 Humans and other living organisms are constantly exposed to a large number of potentially genotoxic environmental chemicals, including the ubiquitous polycyclic aromatic hydrocarbons (PAHs), aromatic amines, allylbenzenes and nitrosamines. 2,3 The PAHs rank very high on the list of environmental chemicals whose toxicity/carcinogenicity holds concern for humans all over the world. The PAHs are formed as products of incomplete pyrolysis of organic materials and during incomplete combustion of fossil fuel. 4 PAHs are also found in substantial quantities in some foods, depending on the method of cooking, preservation and storage, and are detected in a wide range of meats, fish, vegetables and fruits. 2 Since very low amounts of the PAHs are required to initiate tumors in mouse skin, the human population is probably placed at increased risk of developing cancer as a result of significant pollution of the environment by PAHs. 5 The PAHs, e.g., benzo[a]pyrene (BP) and 3-methylcholanthrene (MC), are carcinogenic to laboratory animals, and BP, which is a constituent of cigarette smoke and diesel exhausts, may be involved in the etiology of human cancers associated with exposure to these agents. 6 -8 PAHs ...

Benzo [a]pyrene (BP), a polycylic aromatic hydrocarbon (PAH), is a potent atherogen and carcinogen in laboratory animals. Since genotoxic mechanisms may contribute to the development of atherosclerosis by PAHs, we have tested the hypotheses that: 1) BP induces DNA adducts in mouse aortic smooth muscle cells (SMCs); 2) 3-hydroxybenzo[a]pyrene (3-OH-BP) and benzo [a]pyrene-3,6-quinone (BPQ) are proximate genotoxic metabolites; and 3) cytochrome P4501B1 (CYP1B1) mediates the activation of BP and its metabolites to ultimate genotoxic intermediates. Cultured mouse aortic SMCs were treated with BP, 3-OH-BP, or BPQ for 24 h, and DNA adduct formation was analyzed by 32 P-postlabeling. In some experiments, cells were pretreated with the CYP1B1 inhibitor 1-ethynylpyrene (EP) prior to exposure to BP or its metabolites. BP, 3-OH-BP, and BPQ induced formation of several DNA adducts that were not observed in dimethylsulfoxide-treated cells. Reand cochromatography experiments indicated that 3-OH-BP and BPQ were proximate genotoxic metabolites of BP. DNA adduct formation was strongly inhibited by EP, a specific inhibitor of CYP1B1. BP treatment of SMCs resulted in induction of aryl hydrocarbon hydroxylase (AHH) activity and CYP1B1, but not CYP1A1, apoprotein. EP also blocked AHH induction by BP. In conclusion, the results of this study support the hypothesis that in SMCs, which are target sites for the development of atherosclerosis, the major bioactivation pathway of BP entails CYP1B1-mediated formation of the 3-OH-BP and BPQ, which are proximate genotoxic metabolites that may in turn get transformed to ultimate DNA-binding metabolites, which may contribute to atherogenesis by PAHs.