Richard E. Kouri

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 Ahhi and Ahhn 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.

The quinacrine fluorescence karyotype of Mus musculus and demonstration of strain differences in secondary constrictions

In the mouse, virtually every autosome pair, and the X and Y, was identified by its distinctive fluorescent banding pattern after staining with quinacrine mustard. The karyotype was characterized further by measurement of orcein-stained chromosomes and autoradiographic analysis of terminal DNA replication. The autoradiographic identification of the X and Y chromosomes was confirmed by this technique. Fluorescent karyotype analysis of inbred strains and a series of Fx hybrids involving C57BL/6J revealed a polymorphism, the presence or absence of a prominent secondary constriction on any of four different chromosomes, which was probably inherited in a simple Mendelian fashion.

Correlation of inducibility of aryl hydrocarbon hydroxylase with susceptibility to 3-methylcholanthrene-induced lung cancers

C57BL/6Cum, DBA/2Cum, first filia (F1), and backcross progeny from these 2 parental strains of mice were evaluated for their susceptibility to 3-methylcholanthrene-induced lung cancers. In the crosses among these mice, aryl hydrocarbon hydroxylase (AHH) responsiveness segregated as a single autosomal dominant gene (the Ah locus). AHH responsive mice (Ahb allele) expressed 40-60 units AHH activity/g wet wt liver following intraperitoneal treatment with 3-methylcholanthrene (MCA) compared to AHH non-responsive mice (Ahd allele) which expressed 7-11 units AHH activity/g wet wt liver after MCA treatment. Intratracheal administration of 500 microgram MCA for a total of 4 times at weekly intervals yielded a variety of pulmonary cancers, including squamous cell carcinomas, alveolar adenocarcinomas, and adeno-squamous cell carcinomas among mice that survived 1 year after the carcinogen treatment. The AHH responsive C57BL/6Cum, F1, and C57BL/6Cum X F1 animals were much more susceptible to MCA-induced lung cancers than the AHH non-responsive DBA/2Cum mice. The lung cancers were also not randomly distributed in DBA/2Cum X F1 backcross progeny since significantly more lung cancers were found in AHH-responsive progeny than in AHH non-responsive mice. Data support genetic linkage between susceptibility to MCA-induced lung carcinomas and the Ahb allele.

Increased sister-chromatid exchange in bone-marrow cells of mice exposed to whole cigarette smoke

Using defined cigarette smoke exposure conditions, BC3F1/Cum mice were exposed nose-only to two different types of whole cigarette smoke on a daily basis for 1 week and up to 46 weeks. The number of sister-chromatid exchanges (SCEs) per metaphase was determined in bone-marrow cells. Studies were scheduled so that all cytogenetic observations were made 2-3 days after the last smoke exposure. Exposure to either type of smoke on a daily basis for 1 week or up to 46 weeks resulted in a 2-fold increase in SCEs over sham-exposed control mice. In animals exposed either chronically or for 1 week to either type of smoke, the increase in SCEs persisted for at least 1 week after cessation of smoke exposure. This is the first demonstration of the induction of SCEs in laboratory animals that have been exposed to cigarette smoke in vivo.

Induction of immunotoxicity by polycyclic hydrocarbons: Role of the Ah locus

We have employed the plaque forming cell (PFC) response to sheep erythrocytes as well as lymphocyte proliferation to study the induction of immunotoxicity in AHH-inducible (Ah Locus positive, C57BL/6N; B6C3F1) and AHH non-inducible (Ah Locus negative, DBA2/N) mice following administration of polycyclic aromatic hydrocarbons. When two potent carcinogenic polycyclic hydrocarbons which induce AHH activity, 3-methylcholanthrene (MCA) or 1,2,5,6-dibenzanthracene [DB(a,h)A] were administered IP, immunotoxicity was observed in both AHH-inducible and AHH non-inducible animals. However, the AHH-inducible animals appeared to be more sensitive, and substantial suppression of a PFC response toxicity could be induced with doses as low as 14 mg/kg methylcholanthrene. While suppression of a mitogen response required a dose of 43–125 mg/kg. Administration of the weak carcinogen 1,2,3,4-dibenzanthracene [DB(a,c)A], IP, which similarly induces AHH activity in inducible animals, failed to induce immunotoxicity in either C57B1/6N or DBA/2N mice. In contrast to the results obtained following IP administration, when MCA was administered repeatedly (4X) via an intragastric (IG) route we observed striking immunosuppression of a PFC response in Ah locus negative (DBA/2) animals but minimal effects in Ah locus positive animals (C57B1/6). We finally observed that a single IP dose of MCA (125 mg/kg) to Ah locus positive animals substantially inhibited Natural Killer Cell activity but had more limited effects on the ability of an animal to reject a challenge by an immunogenic syngeneic fibrosarcoma.

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.

The Significance of Aryl Hydrocarbon Hydroxylase Enzyme Systems in the Selection of Model Systems for Respiratory Carcinogenesis

Aryl hydrocarbon hydroxylase (AHH) is a multicomponent, microsomal-bound enzyme system which converts a variety of lipid-soluble compounds to water-soluble forms for subsequent elimination from the body. The enzyme system is inducible by a variety of endogenous and exogenous compounds including steroid hormones, barbiturates, insecticides, polycyclic aromatic hydrocarbons (PAH), and whole cigarette smoke. Recent results have demonstrated that inducibility is host-gene regulated, the inducibility of this enzyme correlates with carcinogenic susceptibility to PAH in animal, and bronchogenic squamous cell carcinoma in humans (probably cigarette smoke induced).

Genetic Differences in Benzo[a]pyrene Carcinogenic Index in Vivo and in Mouse Cytochrome P1 450-Mediated Benzo[a]pyrene Metabolite Binding to DNA in Vitro

Carcinogenic polycyclic hydrocarbons, such as the ubiquitous environmental contaminant BP,1 probably require metabolic activation before initiation of cancer can occur. Boyland first proposed (1) that the initial products of double-bond oxidation are epoxides. When polycyclic hydrocarbon carcinogens are topically applied to mouse skin, covalent binding of the compounds to cellular macromolecules results (2). Subsequently the incubation of BP with microsomes, cofactors, and calf thymus DNA in vitro was shown (3) to produce covalent binding of unknown metabolites to the DNA. It was then appreciated that reactive arene oxides are formed by the microsomal cytochrome P450-mediated monooxygenase systems and that these intermediates can rearrange nonenzymatically to form phenols, be hydrated to form trans-dihydrodiols, and be conjugated with glutathione (4) [cf. refs. 5–8 for reviews].

Assignment of linkage groups VIII and X to chromosomes in Mus musculus and identification of the centromeric end of linkage group I

The mitotic chromosomes in primary embryonic cultured cells of eight translocation stocks of Mus musculus were identified by their distinctive fluorescent banding patterns after staining with quinacrine mustard. By correlation of cytologic and genetic information, linkage group (LG) X was assigned to chromosome 7 and LG VIII to chromosome 5. Several earlier assignments were confirmed: LG I to chromosome 8, LG V to chromosome 2, LG XII to chromosome 19, LG XIII to chromosome 1, and LG XVIII to chromosome 9. In addition, chromosomes 4, 17, and 18 were identified in translocations, but the available genetic data did not permit linkage-group assignments. The centromere of chromosome 8 was located at the nv (Nijmegen waltzer) end of LG I by the two-breakpoint method. The position of the centromere of chromosome 1 at the fz (fuzzy) end of LG XIII and that of chromosome 9 at the nr (nervous) end of LG XVIII were confirmed by the same method.

A method for detecting aryl hydrocarbon hydroxylase activities in cryopreserved human lymphocytes

Aryl hydrocarbon hydroxylase (AHH) activity, NADH-dependent cytochrome c reductase (cyt c) activity, and [3H]thymidine (3H-TdR) incorporation were monitored in human lymphocytes cryopreserved for periods up to 1 year. A standard procedure for freezing, thawing and culturing of these lymphocytes was developed. Kinetics for expression of benz[a]anthracene-(BA)-induced AHH activity, cyt c activity, and 3H-TdR incorporation were similar in both freshly cultured and cryopreserved cells. Lymphocyte samples from 10 individuals were collected once per month over a 3-month period and cells were either cultured at the time of donation or cryopreserved for later assay. Results indicated that the cryopreserved lymphocytes efficiently responded to mitogen activation. The intra-individual variation in AHH activities was reduced in the cryopreserved lymphocytes compared to the freshly cultured cells, and the relative ranking of these individuals in terms of their AHH activities remained constant for both fresh and cryopreserved samples. Cryopreservation seems to offer significant advantages over the freshly cultured lymphocytes because it allows for lymphocyte samples to be collected in diverse geological locations and over extended periods of time and yet permits for the culture and assay of all the cell samples at exactly the same time.