Relationship between induction of aryl hydrocarbon hydroxylase and de novo synthesis of cytochrome P-448 (P1-450) in mice

Phenobarbital, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT), benzpyrene, 3-methylcholanthrene (3-MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were administered i.p. for 1 or 3 days to genetically “responsive” (C57BL/6J) and genetically “non-responsive” (DBA/2J) mice. 3-MC or benzpyrene stimulated aryl hydrocarbon hydroxylase (AHH) activity in C57BL/6J (B6) mice but not in DBA/2J (D2) mice. TCDD induced AHH activity in both B6 and D2 mice. Time-course studies showed that in the first 12 h after a single injection of 3-MC to B6 mice there was no shift in the reduced cytochrome P-450-CO complex absorption spectra from 450 to 448 nm, although AHH activity increased 4–5 times over (above) that of the control group. The relationship between induction of AHH activity by polycyclic hydrocarbons in B6 mice and the concomitant synthesis of cytochrome P-448 is discussed.     

Simple vs. complex inheritance of inducible aryl hydrocarbon hydroxylase in mouse tissues

The genetics of induction of hepatic and lung aryl hydrocarbon hydroxylase (AHH) have been studied in Af/Ki and AKR/Ki mice and in their F1 and F2 progeny after administration of 3-methylcholanthrene (3MC). Furthermore, the induction of AHH was investigated using the fetal liver explant model system with 3MC, trans-1,2-dihydroxy-3MC, and 4'-bromoflavone as the inducers. The results obtained with the above strains were contrasted with those from the C57BL/6Ki, DBA/2+Ki, and their crosses. The present investigation revealed a complex pattern of inheritance of basal and inducible AHH in lung and liver of AKR/Ki and Af/Ki, with a poor correlation between lung and liver. Hepatic AHH was not fully inducible in the F1 hybrids, while the frequency distribution function in the F2 mice was suggestive of more than two distinct classes.     

Studies on pulmonary aryl hydrocarbon hydroxylase activity in inbred strains of mice.

Pulmonary and hepatic levels of aryl hydrocarbon hydroxylase (AHH) were studied in inbred strains of mice following intratracheal (i.t.) instillation of 3-methylcholanthrene (MCA). I.t. instillation of 188 mug MCA in sterile 0.2% gelatin in saline resulted in preferential induction of pulmonary AHH. After treatment with this dose of MCA, the pulmonary AHH levels of strains C57BL/6Cum, C57BL/6J, BALB/cMai, C3H/fMai, and C57L/J were observed to be induced within 24 h after treatment. Strains DBA/2Cum, AKR/J, SJL/J, DBA/2J and RF/J expressed no such increase. At a dose of 500 mug MCA, the pulmonary tissue of DBA/2 mice did express a 4-fold increase. This increase in AHH was determined to be quite different from the increase observed in C57BL/6 mice by: (1) specific activity of the enzymes, (2) genetic regulation, (3) susceptibility to inhibition by 7,8-benzoflavone, and (4) spectral properties of the associated cytochromes. It was of major importance that induction of pulmonary AHH was observed to be regulated by a single dominant gene in crosses involving the C57BL/6Cum and DBA/2Cum strains of mice. Results were discussed with the view in mind that these genetically regulated levels of AHH may play a role in susceptibility to cancers induced by polycyclic aromatic hydrocarbon carcinogens.     

Genetic regulation of UDP-glucuronosyltransferase induction by polycyclic aromatic compounds in mice. Co-segregation with aryl hydrocarbon (benzo(alpha)pyrene) hydroxylase induction.

Induction of hepatic 4-methylumbelliferone UDP-glucuronosyltransferase (EC 2.4.1.17) by polycyclic aromatic compounds, such as 3-methylcholanthrene or beta-naphthoflavone, occurs in C57BL/6N, A/J, PL/J, C3HeB/FeJ, and BALB/cJ but not in DBA/2N, AU/SsJ, AKR/J, or RF/J inbred strains of mice. This pattern of five responsive and five nonresponsive mouse strains parallels that of the Ah locus, which controls the induction of aryl hydrocarbon (benzo[alpha]pyrene) hydroxylase (EC 1.14.14.2). Induction of the transferase is maximal in C57BL/6N mice with 200 mg of 3-methylcholanthrene/kg body weight; no induction occurs in nonresponsive DBA/2N mice even at a dose of 400 mg/kg. The rise of inducible transferase activity lags 1 or more days behind the rise of inducible hydroxylase activity and peaks 5 days after a single dose of 3-methylcholanthrene. In offspring from the appropriate backcrosses and intercross between C57BL/6N and DBA/2N parent strains, the genetic expression of 3-methylcholanthrene-inducible transferase activity is inherited as an additive (co-dominant) trait. This expression differs distinctly from that of the inducible hydroxylase activity, which is inherited almost exclusively as a single autosomal dominant trait in these same animals. The more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin induces the transferase more than 3-fold in C57BL/6N mice and less than 2-fold in DBA/2N mice, whereas the hydroxylase is induced equally (about 8-fold) in both strains. A dose of 3-methylcholanthrene given 3 days after 2,3,7,8-tetrachlorodibenzo-p-dioxin, at a time when hydroxylase induction in both strains is very high, does not enhance the rise in inducible transferase activity seen in C57BL/6N or DBA/2N mice which have received 2,3,7,8-tetrachlorodibenzo-p-dioxin alone. These data indicate that (a) the inducibility of two metabolically coordinated membrane-bound enzyme activities may be regulated by a single genetic locus, and (b) although the hydroxylase can be fully induced in the nonresponsive DBA/2N strain by 2,3,7,8-tetrachlorodibenzo-p-dioxin prior to 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene treatment, metabolites of the 3-methylcholanthrene, presumably present in the liver, are incapable of inducing further the transferase activity. The difference in sensitivity between 3-methylcholanthrene and the more potent inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin for both the hydroxylase and the transferase activities suggests the possibility of a common receptor in regulating both enzyme induction processes.     

Selection of inducers: An important factor in characterizing genetic differences to induction of aryl hydrocarbon hydroxylase in strains of mice

The effect of various microsomal enzyme inducers such as DDT, benzpyrene, 3-MC, TCDD or phenobarbital on liver microsomal mixed-function oxidases and cytochrome P450 content in mice genetically responsive (C57B1/6J) and resistant (DBA/2J) to induction of aryl hydrocarbon hydroxylase (AHH) was studied. 3-MC and benzpyrene administration stimulated liver AHH activity 6–8 fold in C57B1/6J mice but had no effect in DBA/2J mice. However, intraperitoneal administration of TCDD increased AHH activity in both C57BL/6J and DBA/2J mice. This increase was accompanied by shift in the peak of cytochrome P450 difference spectrum from 450 to 448 nm. It is concluded that genetic resistance to AHH stimulation in DBA/2J mice is influenced by the type of inducer used.     

Monoclonal antibody-directed phenotyping of cytochrome P-450-dependent aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase in mammalian tissues

The distribution of cytochromes P-450 that catalyze aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase were studied with monoclonal antibody (MAb) 1-7-1 which completely inhibits these activities of a purified 3-methylcholanthrene-induced rat liver cytochrome P-450. The degree of inhibition by MAb 1-7-1 quantitatively assesses the contribution of different cytochromes P-450 in the liver, lung, and kidney microsomes from untreated, 3-methylcholanthrene- and phenobarbital (PB)-treated rats, mice, guinea pigs, and hamsters. Enzyme sensitivity to MAb 1-7-1 inhibition defines two types of cytochrome P-450 contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase. The MAb 1-7-1-sensitive cytochrome P-450 is a major contributor to aryl hydrocarbon hydroxylase in rat liver, lung, and kidney of 3-methylcholanthrene-treated rats, C57BL/6 mice, guinea pigs, and hamsters; this type is also present in lesser amounts in the extrahepatic tissues of the control and PB-treated animals, and in the lungs of the relatively "noninducible" DBA/2 mice treated with 3-methylcholanthrene. This form however makes little or no contribution to liver aryl hydrocarbon hydroxylase of control or PB-treated animals. 7-Ethoxycoumarin O-deethylase is also a function of both the MAb 1-7-1-sensitive and insensitive classes of cytochrome P-450. The ratio of the classes contributing to aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-deethylase differs in the various tissues and species and after inducer treatment. All of the 7-ethoxycoumarin O-deethylase activity in guinea pigs and hamsters is a function of cytochromes P-450 different than the MAb 1-7-1-sensitive cytochrome P-450 responsible for aryl hydrocarbon hydroxylase activity. Thus, the MAb 1-7-1 antigenically defines the type of cytochromes P-450 contributing to each reaction. Cytochromes P-450 can be viewed as paradigmatic for enzyme systems in which the nature and amount of product is regulated by multiple isoenzymic forms. Analyses using monoclonal antibodies to specific isoenzymes may thus have broad application to a variety of other complex systems which are composed of multiple isoenzymes.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Comparison of the in vitro mutagenicity and metabolism of dimethylnitrosamine and benzo[a]pyrene in tissues from inbred mice treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls.

Homogenates of liver, lung, kidney, stomach, small intestine and colon from 8 strains of mice were compared for their ability to metabolize benzo[a]pyrene (BP) and dimethylnitrosamine (DMN) to mutagens. Females of strains CF1, AKR/J, AU/SsJ, DBA/2J, SWR/J, A/J, C3H/HeJ, and C57BL/6J were either untreated or received phenobarbital (PB), 3-methylcholanthrene (MC) or polychlorinated biphenyls (AR) to induce drug-metabolizing enzymes. The effects of these drugs on organ weight and on the amounts of DNA, S-10 protein, and microsomal protein per unit weight of tissue are reported. Salmonella typhimurium TA92 and TA98 were used as indicators of the formation of mutagens. For each organ there was an optimal balance between amount of tissue homogenate and concentration of test compound for maximal yield of revertants. A sensitive radiometric assay of DMN demethylase (DMND) is described which permits measurement of the enzyme in liver, lung and kidney. DMN at 1 mM is used as substrate. Aryl hydrocarbon hydroxylase (AHH) was measured in all tissue using BP as substrate. AR and MC are very good inducers of AHH activity in livers of mice classified as aromatic hydrocarbon responsive, but not in those classified as hydrocarbon nonresponsive. Responsiveness is strain-specific and genetically regulated. Metabolism of BP to mutagens by liver homogenates was correlated with extent of AHH induction. This dimorphism of response of AHH to inducers was present, but less pronounced, in non-hepatic tissues. Basal activities of AHH and DMND were correlated in livers and lungs from untreated mice. DMND activities were increased less than 2-fold by PB, MC or AR treatments. Metabolism of DMN to mutagens was not closely correlated with DMND activities. Strain of mouse, type of tissue and test substance are important variables in assessing the potential effect of microsomal enzyme-inducing agents on the metabolism of mutagenic substances.     

The genetics of aryl hydrocarbon hydroxylase induction in mice: A single gene difference between C57BL/6J and DBA/2J

Twenty-one inbred strains of mice were surveyed for inducibility of hepatic aryl hydrocarbon hydroxylase (AHH) activity by the carcinogen 3-methylcholanthrene (MC). In 11 strains given MC, AHH activity increased 1.3- to 5-fold (inducible), whereas ten strains responded with a less than 0.5-fold increase (noninducible). Neither the inducible nor the noninducible class was homogeneous, and in each considerable variation was found in both the basal activity of AHH and the response to MC. Strains DBA/2J and C57BL/6J were chosen to represent the noninducible and inducible classes, respectively. In the crosses (C57BL/6 × DBA/2)F₁ × DBA/2 and (C57BL/6 × DBA/2)F₂, inducibility segregated as a single autosomal dominant gene. The gene symbols Ahh ⁱ and Ahh ⁿ are proposed for the alleles present in C57BL/6J and DBA/2J, respectively. No genetic linkage was found between the Ahh locus and the following loci: b, d, Es-1, Es-3, Gpd-1, Hbb, Id-1, Pgm-1, and sex. Some implications of this work in the study of mammalian enzyme induction and chemically induced carcinogenesis are discussed. There is a positive correlation between AHH inducibility and the development of an inflammatory response to the topical application of the carcinogen 7,12-dimethylbenzanthracene.     

Genetics of aryl hydrocarbon hydroxylase induction in mice: Response of the lung to cigarette smoke and 3-methylcholanthrene

When mice from different inbred strains are injected intraperitoneally with 3-methylcholanthrene (MC), the activity of aryl hydrocarbon hydroxylase (AHH) rapidly increases in livers of some strains but not others. AHH plays a role in the metabolism of polycyclic hydrocarbons. Alleles at a small number of loci account for most of the variation in inducibility of hepatic AHH among mice, when MC is used as the inducing agent. Cigarette smoke is a common source of carcinogenic polycyclic hydrocarbons in the environment. Since some of the hydrocarbons in cigarette smoke are metabolized by AHH, the activity of AHH in tissues may affect the carcinogenicity of smoke in those tissues. The purpose of these experiments was to see whether induction of AHH in lung in response to cigarette smoke is regulated by the same genes that regulate induction of AHH in liver in response to MC. Mouse strains AKR/J and C57L/J and six recombinant inbred (RI) lines derived from them were tested for the response of AHH in lung and liver to parenteral MC or inhalation of cigarette smoke. Inducibility (the ratio of MC-induced AHH activities to basal AHH activities) in liver from MC-treated RI lines is bimodal and compatible with Mendelian segregation of genes at a small number of loci. Increased activities of AHH in MC-treated liver are associated with increased ability to metabolize BP and whole smoke condensates to mutagens detected by Salmonella typhimurium TA1538. Inducibility of AHH in lung in response to MC is not bimodal, and no definite conclusion about the number of loci can be made. When actual levels of AHH activity are considered, following the administration of MC as inducing agent, there is a correlation (r=0.89, p<0.01) between AHH levels in liver and lung, suggesting that some genes affecting liver also affect lung. Basal and MC-induced AHH levels in lung are also correlated (r=0.86, p<0.01). Mice with high basal activities have two to threefold higher levels of AHH after MC treatment than do mice with low basal activities. Induction of AHH in pulmonary tissues occurs in all mice after either parenteral MC or smoke inhalation. In contrast to MC treatment, AHH activities in lungs following smoke inhalation are not correlated with AHH levels in liver after MC (r=0.49) and are only weakly correlated with basal (r=0.66, 0.05相似文献   

Cutaneous metabolism of benzo[a]pyrene: comparative studies in C57BL/6N and DBA/2N mice and neonatal Sprague--Dawley rats

The metabolism of the polycyclic aromatic hydrocarbon (PAH) carcinogen benzo[a]pyrene (BaP) was studied using microsomes prepared from the skin of the mouse and rat. Topical application of the polychlorinated biphenyl (PCB) Aroclor 1254 or the PAH 3-methylcholanthrene (3-MC) to the skin of the C57BL/6N and DBA/2N mouse and the Sprague-Dawley rat caused statistically significant enhancement of cutaneous microsomal aryl hydrocarbon hydroxylase (AHH) activity in each animal. PCB was a more potent inducer of the enzyme than was 3-MC. BaP metabolism by skin microsomes from the same animals was assessed using high performance liquid chromatography (HPLC). The skin of untreated animals metabolized BaP into 9,10-, 7,8- and 4,5-dihydrodiols, phenols and quinones. Skin application of PCB caused greater than 16–18-fold enhancement of BaP metabolism in the C57BL/6N mouse and the rat and 2–5-fold enhancement in the DBA/2N mouse. Skin application of 3-MC enhanced BaP metabolism 2–8-fold in the C57BL/6N mouse and 5–10-fold in the rat and had no effect in the DBA/2N mouse. The formation of procarcinogenic metabolite BaP-7, 8-diol was greatly enhanced (4–12-fold) by treatment with the PCB and 3-MC in the tumor susceptible C57BL/6N mouse and in the tumor-resistant neonatal Sprague-Dawley rat. In contrast, the formation of BaP-7,8-diol was either slightly enhanced (2-fold) or unaffected by treatment with the PCB or 3-MC in the tumor-resistant DBA/2N mouse. Our data indicate that neither the patterns of metabolism nor the amount of BaP-7,8-diol formation in the skin are reliable predictors of tumor susceptibility to the PAH in rodent skin.     

Detection of aryl hydrocarbon hydroxylase activity in normal and neoplastic human breast epithelium

Studies were conducted to determine whether normal and/or neo-plastic (MCF-7) human breast epithelial cells contain the microsomal aryl hydrocarbon hydroxylase (AHH) which catalyses the conversion of polycyclic aromatic hydrocarbons (PAH) to carcinogenic intermediates. Low constitutive levels of AHH activity were found in homogenates of both normal human breast epithelial and MCF-7 cells. The addition of 7,12-dimethylbenz(a)anthracene (DMBA) to the culture medium of either cell type significantly increased AHH activity. Peak induction of hydroxylase activity occurred following the $\text{in}$$\text{vitro}$ addition of 10 μM DMBA. A time course of DMBA-induced AHH activity in both normal human breast epithelium and MCF-7 cells revealed maximal induction 16 hr after 10 μM DMBA was added to the culture medium. Benzo(a)pyrene (BP), 3-methylcholanthrene (MCA) and benz(a)anthracene (BA) also induced AHH activity in normal and MCF-7 cells. For example, the addition of 10 μM BP to the culture medium of either normal human breast epithelial or MCF-7 cells for 16 hr increased AHH activity 13.8 and 65.3-fold, respectively. For all PAH, the magnitude of AHH induction was substantially greater in MCF-7 than normal breast epithelial cells. Finally, α-naphthoflavone inhibited BA-induced AHH activity in MCF-7 cells. The study demonstrates the presence of a PAH-inducible AHH enzyme(s) in normal human breast epithelial cells grown in primary culture and in the human breast tumor cell line, MCF-7.     

Expression of aromatic polycyclic hydrocarbon-induced monooxygenase (aryl hydrocarbon hydroxylase) in man x mouse hybrids is associated with human chromosome 2

Summary Polycyclic aromatic hydrocarbon-inducible monooxygenase directed toward the substrate benzo(a)pyrene, i.e., aryl hydrocarbon hydroxylase, was monitored in cell hybrids formed from mouse RAG cells and several human fibroblasts lines. In RAG cells no aryl hydrocarbon hydroxylase activity was detectable; however, these cells exhibited relatively high levels of NADPH cytochrome C (P-450) reductase (EC. 1.6.2.4). In 12 hybrids lines, induced aryl hydrocarbon hydroxylase segregated with human chromosome 2. The results indicate that the structural gene of the polycyclic aromatic hydrocarbon-inducible monooxygenase or gene(s) involved in the induction of the enzyme is located on human chromosome 2.Abbreviations AHH aryl hydrocarbon hydroxylase - IDH isocitrate dehydrogenase - MDH malate dehydrogenase - PAH polycyclic aromatic hydrocarbons     

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.     

The murine aromatic hydrocarbon responsiveness locus: a comparison of receptor levels and several inducible enzyme activities among recombinant inbred lines

The aromatic hydrocarbon responsiveness (Ah) locus has been correlated with genetic differences in the risk of drug toxicity, teratogenesis, chemical carcinogenesis, and mutagenesis. Hepatic cytosolic Ah receptor levels, 2-amino-5-chlorobenzoxazole (zoxazolamine) paralysis time following beta-naphthoflavone treatment and aryl hydrocarbon hydroxylase (AHH3, acetanilide 4-hydroxylase (Ac4H), and NAD(P)H:menadione oxidoreductase (NMOR)4, induction by 3-methylcholanthrene were studied in (a) the progenitors C57BL/6J (Ahb/Ahb) and DBA/2J (Ahd/Ahd) and 25 BXD recombinant inbred lines, (b) the progenitors C57BL/6N and C3H/HeN and 14 B6NXC3N recombinant inbred lines, and (c) the progenitors C57BL/6J and C3H/HeJ and 12 BXH recombinant inbred lines. The Ahb phenotype exhibits greater than 5 femtomole receptor/mg of cytosolic protein, less than or equal to 15 minutes zoxazolamine paralysis time, and twofold to 15-fold induction of these three hepatic enzyme activities; the Ahd phenotype exhibits less than or equal to 2 fmol receptor/mg protein, greater than 15 minutes zoxazolamine paralysis time, and less than 30% induction of these three activities. Among the BXD lines but especially among the B6NXC3N and BXH lines, high frequencies of recombination were found; the phenotype of each of the five parameters did not segregate with the phenotype of each of the other parameters in four or more recombinant lines. This report shows for the first time that AHH induction by 3-methylcholanthrene can occur in the Ahd phenotype mouse. These data underline the complexity of this genetic system when genes from C57BL/6 and DBA/2 are combined and particularly when genes from C57BL/6 and C3H/He inbred mouse strains are combined.     

Induction of cytochrome P-450IA1 in fetal rat liver by a single dose of 3-methylcholanthrene.

Pregnant Sprague-Dawley rats were treated with a single ip dose of either olive oil or 40 mg/kg of 3-methylcholanthrene on gestation day 20 and sacrificed at various times after injection. Determination of aryl hydrocarbon hydroxylase activity 24 hr after injection revealed that treatment with 3-methylcholanthrene resulted in a 10.5-fold stimulation of enzymatic activity in liver 800 x g supernatants. Western blot analysis with monoclonal antibody 1-7-1 confirmed these results and demonstrated the presence of a 3-methylcholanthrene-inducible P-450 isozyme. Using Northern and slot blot techniques, the induction of steady-state levels of CYPIA1 RNA was shown to occur as early as 4 hr following 3-methylcholanthrene injection. CYPIA1 RNA levels were induced 31.6-fold over values obtained from oil-treated tissues at this time. This appears to be the optimal time to study changes in the levels of CYPIA1 RNA gene expression in the fetus following transplacental exposure to polycyclic aromatic hydrocarbons. By 12 to 24 hr postinjection, the induction of CYPIA1 RNA levels declined to 3.5- to 8.5-fold above control values. These results demonstrate that the kinetics of induction of the CYPIA1 gene during the fetal period differed from that seen in adults.     

Induction of aryl hydrocarbon hydroxylase in human lymphocytes and pulmonary alveolar macrophages--a comparison

Exposure of animals to cigarette smoke causes an increase in the levels of aryl hydrocarbon hydroxylase (AHH) in various tissues. The innate capacity for enzyme induction is genetically determined but the extent of induction and AHH levels in various tissues may vary. AHH levels in human pulmonary alveolar macrophages (PAMs) were determined and AHH inducibility of cultured lymphocytes from corresponding volunteers was determined. The inducibility by 3-methylcholanthrene of cultured lymphocytes was similar in both smokers and nonsmokers, ranging from 0.2 – 4.2 fold induction. AHH levels in nonsmoker PAMs was 0 – 0.020 units and in smokers was 0.032 – 0.253 units. The correlation of AHH activity in PAMs with lymphocyte inducibility was significant in both nonsmokers and smokers. The regression of AHH in PAMs as compared with AHH inducibility in lymphocytes was 8.5 fold higher in smokers than nonsmokers, reflecting the induction of AHH in PAMs by smoking.     

Benzo[a]pyrene-DNA-adducts and monooxygenase activities in mice treated with benzo[a]pyrene, cigarette smoke or cigarette smoke condensate

Synchronous fluorescence spectrophotometry (SFS), developed to study benzo[a]pyrene-7,8-diol-9,10-epoxide(BPDE)-DNA, was used to measure the in vivo formation of DNA-adducts in genetically responsive C57BL/6 (B6) and non-responsive DBA/2 (D2) mice. Treatment with cigarette smoke by inhalation for 3-16 days, or i.p. injection of cigarette smoke condensate or neutral fraction did not lead to detectable levels of BPDE-DNA-adducts in either lungs or liver, although aryl hydrocarbon hydroxylase (AHH) activity, an indicator of benzo[a]pyrene (BP) metabolism, was clearly induced in lungs of B6 mouse. A dose-dependent amount of BPDE-DNA-adducts in lung and somewhat less in liver was found after i.p. injection with BP (20-80 mg/kg). Mice treated with vehicle or 4 mg/kg of BP were negative for adducts by SFS. In B6 mice AHH was induced both in lungs and livers while there was no AHH induction in D2 mice although the levels of BPDE-DNA-adducts were somewhat higher than in B6 mice. Thus, no clear correlation seems to exist between AHH activity and the formation of BPDE-DNA-adducts. Also, according to our results SFS can be used to quantitate adduct-formation in in vivo animal studies.     

Genetically mediated induction of drug-metabolizing enzymes associated with congenital defects in the mouse.

Various polycyclic aromatic compounds induce certain monooxygenase activities, including aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.2), and cytochrome P1-450 in the liver and many nonhepatic tissues of the mouse. This induction process is controlled by the Ah locus. Genetic differences that have been shown in the past to be associated with the Ah locus include an increased susceptibility to chemical carcinogenesis, mutagenicity in vitro, and drug toxicity--manifested as hepatic necrosis, aplastic anemia, or shortened survival time. Pregnant mice received a single injection of 3-methylcholanthrene or 7,12-dimethylbenz[a] anthracene between day 5 and day 13 of gestation, and the uterine contents were examined on day 18. Striking increases were observed in the incidence of MC-1 and DMBA-induced resorptions and congenital malformations in the aromatic hydrocarbon "responsive" C57BL/6N inbred strain, and of DMBA-induced resorptions in the "responsive" C3H/HeN and BALB/cAnN strains--when compared with the similarly treated genetically "nonresponsive" AKR/N strain. These data suggest but do not prove that an association exists between the Ah locus and developmental toxicity, i.e., teratogenesis. Although numerous teratogenic differences among inbred mouse strains have previously reported, this study is unique in that the genetic differences in teratogenicity observed were predicted in advance on the basis of known differences among these strains in polycyclic hydrocarbon metabolism regulated by the Ah locus.     

Announcement

Aryl hydrocarbon hydroxylase (AHH) was present in explant cultures of human prostate obtained from surgery of benign prostatic hyperplasia and was inducible by benz[a]anthracene (BA). The induction of AHH ranged from 14- to 150-fold when compared with control values and 10-fold variation of AHH inducibility among individuals was observed. Epithelial cells grown from human prostate tissue also contained measurable AHH activity and AHH was inducible by BA and 7,12-dimethylbenz[a]anthracene (DMBA). Inducibility of AHH by BA ranged from 2- to 63-fold. The inducibility of AHH by DMBA was always less than that by BA. In cells treated with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), there were no changes in AHH activity. These findings support the view that the human prostate is susceptible to environmental polycyclic hydrocarbon carcinogens and that environmental and occupational factors might contribute to the etiology of human prostatic carcinoma.