Mouse Models Of The Arylhydrocarbon Receptor And Nacetyltransferase Polymorphisms

Interest in the arylhydrocarbon receptor and N-acetyltransferase polymorphisms stems from their roles in toxicity from environmental chemicals. Variant forms of the Ah-receptor protein and of N-acetyltransferase, a drug-metabolizing enzyme protein, are encoded by the responsible genes. The polymorphisms at these loci influence individual variations at pharmacodynamic and pharmacokinetic levels of drug response, respectively. The Ah locus is located on mouse chromosome 12 and the N-acetyltransferase loci are on mouse chromosome 8; hence they are not linked. The nomenclatures for the Ah locus, Ahr (formerly Ah), and for N-acetyltransferase, Nat*, are used in this discussion.

Arylhydrocarbon Receptor Polymorphism

The Ahr receptor is a ligand-activated transcription factor that regulates a large number of biological responses to planar aromatic hydrocarbons such as ben-zo[a]pyrene, 3-methylcholanthrene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). It is best known for its role in mediating biological responses to ha-logenated dioxins and related toxic and carcinogenic environmental chemicals. A series of observations in rats, mice, and various other species had indicated that 3-methylcholanthrene is a potent inducer (i.e., causes increased synthesis) of arylhydrocarbon hydroxylase as well as other xenobiotic metabolizing enzymes. A mouse strain survey revealed the locus was polymorphic, that is some mouse strains such as B6 and C3H were responsive to induction while other strains such as DBA and AKR were unresponsive. Responsiveness segregated as a simple autosomal dominant Mendelian character and the responsible locus was named the Ah (renamed Ahr) locus. Strains carrying the "responsive" allele were designated Ahrb (from B6 mice) and the "nonresponsive" allele was designated Ahrd (from DBA mice).19,20

Experimental proof that the Ahr locus encodes a receptor came from efforts of many laboratories that began in the 1960s and spanned more than two decades. Formal proof of this concept is based on the demonstration of high- and low-affinity binding sites that segregate with responsiveness and nonresponsiveness, and the demonstration that hydrocarbon ligands cause an increased affinity of the receptor for DNA and its redistribution from the cytosolic compartment of the cell to the nuclear compartment. Further investigation revealed that responsive mice possess receptors having apparent molecular weights of 94 kDa (Ahrb-1), 104 kDa (Ahr^-2), and 105 kDa (Ahrb~ 3), whereas nonresponsive mice possess receptors having a molecular weight of 104 kDa (Ahrd).21,22

The concept that the Ah receptor is a ligand-activated transcription factor evolved from experiments in mice that demonstrated that induction of arylhy-drocarbon hydroxylase activity was blocked by inhibitors of transcription or translation, and that on binding the agonist the Ah receptor develops a high affinity for the cell nucleus and for DNA. Agonist-dependent up-regulation of the receptor comes about because of increased transcriptional initiation. Evidence from deletion analysis indicated the presence of regulatory domains located in the 5' region of genes that respond to ligand (TCDD)-induced induction, such as the P450 gene, Cyp1A1. The mouse TCDD-responsive domain possesses the characteristics of a typical enhancer element, that is, it activates transcription at a distance from the promoter in an orientation-independent manner. For Cyp1A1, enhancer activity is the sum of effects of at least six distinct responsive elements acting independently in some cases and cooperatively in others. These elements are designated by different groups as dioxin-responsive, or xenobiotic-responsive, or Ah-responsive receptor elements and are abbreviated as DREs,

XREs, and AHREs, respectively. Direct sequencing, footprinting, and mutational analysis of these elements defined the core recognition sequence, GCGTG, as that essential for receptor interaction and enhancer function; outside this core the sequence TNGCGTG corresponds to a site with the highest affinity.

A number of genes that may be upregulated in response to arylhydrocarbons such as TCDD have been identified and referred to as members of the "Ah gene battery.'' As a group, they are characterized by rapid increases in protein and mRNA levels after exposure to the agonist. For example, Ah receptor DRE-mediated increases in Cyp1A1 mRNA are observed within 30 minutes of exposure to TCDD. Genes for a number of drug-metabolizing enzymes are assigned to this gene battery, and several additional genes as yet to be identified appear to be regulated by the Ah receptor gene.

Somatic cell genetics carried out on mouse-derived cell lines proved to be a fruitful way to identify additional genes involved in the Ah receptor signaling pathways that lead to induction of arylhydrocarbon hydroxylase activity (largely Cyp1A1 activity). Complementation studies have revealed that mutations at four distinct loci can generate resistant phenotypes: (1) mutations that inactivate the responsive gene, Cyp1A1, (2) those that have approximately 10-fold lower cytosolic receptor concentrations, (3) those that have wild-type receptor concentrations but the receptor fails to show increased nuclear affinity on agonist binding, and (4) those that appear to exhibit both of the latter defects—i.e., a low receptor number and decreased nuclear affinity of the ligand-bound receptor. Rescuing experiments of the decreased nuclear affinity mutants demonstrated that the Ah receptor required a dimeric partner to bind the DREs. This protein became known as ARNT because its absence correlated with decreased affinity of the Ah receptor for the nuclear compartment. Apart from their value in identifying additional genes involved in Ah receptor pathways, the complementation pattern of somatic cell mutants and the importance of the ARNT protein were some of the first indicators to distinguish the Ah receptor from the steroid/thyroid receptor supergene family and prove it to be a unique signaling molecule.

The Ah receptor and ARNT molecules had sequence identity with the Per and Sim proteins of Drosophila, proteins of a family of transmission factors. This family is characterized by a motif referred to as the "PAS" domain, which harbors sequences in the Ah receptor involved in the formation of a hydrophilic pocket that binds the arylhydrocarbon agonist. Adjacent to the PAS domain is a basic/helix-loop-helix (bHLH) domain that is believed to mediate hetero-dimerization and sequence-specific DNA-binding properties. The domain map is consistent with the observation that the Ah receptor and ARNT proteins are heterodimeric partners that activate gene expression. A hypothetical model of the receptor based on putative functional domains observed in mice has been proposed by Hollie Swanson and Christopher Bradfield.23 Older versions of a molecular model of the mechanism by the Ah receptor transduce the signal of arylhydrocarbon agonists and thus have been revised because they were based incorrectly on the premise that the Ah receptor was a member of the steroid/ thyroxine gene family.

Several studies have been performed in congenic mouse strains differing in their Ahr receptor responsiveness. The acute toxicity of TCDD, a potent inducer of the Ah receptor, has been examined in male mice differing only at the Ahr locus. In a study by Birnbaum and colleagues, the LD50 values were 159 and 3351 mg/kg body weight for responders and nonresponders, respectively, although the mean time of death (22 days) was independent of dose and Ahr receptor genotype.24 Among various anatomical and biochemical signs of dose-related acute toxicity that were observed, necrosis of germinal epithelium in the testes occurred at doses 8-24 times greater in nonresponsive than in responsive mice. The spectrum of toxicity was found to be dependent on the Ahr genotype, but the relative doses required to bring about acute responses to TCDD are much greater in congenic mice homozygous for the nonresponsive allele than for mice homozygous for the responsive Ahr allele.

The pathogenesis of hexachlorobenzene-induced porphyria was investigated in female B6.Ahrb (responsive) and B6.Ahrd (nonresponsive) congenic strains.25 TCDD led to urinary excretion of porphyrins that was 200 times greater in untreated responsive control mice compared to only 6 times greater in untreated nonresponsive control females. The hepatic accumulation of hexachlorobenzene was also greater in responsive than in nonresponsive mice and was associated with greater hepatic lipid levels in the former strain. These findings as well as other biochemical indices indicated that the Ahr locus influences the susceptibility of mice to hexachlorobenzene-induced porphyria, and further studies showed that specific P450 isoforms that are members of the Ahr gene battery are likely causative factors in the development of this disorder.

The effect of 3-methylcholanthrene on atherosclerosis has also been examined in congenic mouse strains that differ in Ah responsiveness.26 The effect of 3-methylcholanthrene was found to be greater in responsive mice receiving an atherogenic diet than in nonresponsive mice treated similarly. Thus, exposure to 3-methylcholanthrene increased the area of atherosclerotic lesions in both con-genic strains, but the magnitude of the increase was significantly greater in Ahr-responsive than nonresponsive mice even though the high-density lipoprotein levels were not significantly altered by such treatment or by the Ahr receptor genotype. The study of the F1 progeny of responsive (AKXL-38a) and nonresponsive (AKXL-38) mice backcrossed to the nonresponsive parent revealed that increased susceptibility to 3-methylcholanthrene-induced atherosclerosis segregated with the Ahr receptor locus.

A developmental role for the Ahr locus was indicated by the observation that mice congenic to the C57BK/6 strain harboring a null allele show a portocaval vascular shunt throughout life.27 Three-dimensional (3D) visualization at various developmental times indicated that the shunt is an embryonic remnant acquired before birth. The ontogeny of the shunt plus its 3D position suggested that the shunt is due to a patent ductus arteriosus. During the first 48 hours, most major hepatic veins including the portal and umbilical veins usually decrease in diameter but do not change in Ahr null mice. In searching for its physiological cause, it appears that failure of the ductus to close may be a consequence of increased blood pressure or a failure in vasoconstriction in the developing liver.

Acetylation Polymorphism

The hereditary variability in the acetylation of an array of chemicals with ar-ylamine and hydrazine moieties is a well-known trait that occurs in humans (see Appendix A), mice, and several other mammalian laboratory animal species. This trait is determined by significant differences in N-acetyltransferase (NAT2) activity in liver and several other tissues, and it is referred to as the N-acetyl-transferase polymorphism.

A survey of inbred mouse strains identified 17 with rapid acetylator pheno-types and 3 with slow phenotypes. Studies in two representative strains, C57BL/6 mice representing rapid acetylation and A/J mice (henceforth referred to as B6 and A mice, respectively) representing slow acetylation, have repeatedly demonstrated that the polymorphism observed in Nat activity in the liver of mice also occurs in kidney, urinary bladder, blood, colon, and other tissues. These mice were chosen because they differ in many physiological, anatomical, behavioral, and oncological traits including important models of birth defects and adult diseases in humans.28 Further studies of A and B6 mice by standard intercross and backcross matings demonstrated that a single gene with two major codom-inant alleles accounted for the differences in Nat activity. Studies in recombinant inbred strains derived from B6 and A parental mice confirmed the Mendelian inheritance of NAT activity and also revealed significant background gene effects on the differential Nat allele. The metabolic, molecular genetic, and toxicological aspects of the acetylation polymorphism summarized below are reviewed by Levy and colleagues.29

Biochemical studies with prototypical substrates for Nat, p-aminobenzoate (PABA), and the arylamine carcinogen, 2-aminofluorene (AF), showed tissue-specific variations in Nat activity ranging from 1.5-fold to 20-fold in vitro for liver and 21 other tissues. In agreement with these observations, the elimination of AF from blood was two to three times higher for B6 than A mice and for B6 and A hepatocytes isolated in primary culture. On the other hand, no differences in rates of isoniazid N-acetylation in vitro or in vivo were detected in urine of B6 and A mice.

The large difference in blood PABA N-acetylating activity between B6 and A phenotypes (up to 20-fold), and the ability to discriminate heterozygote animals from homozygous rapid and slow phenotypes by assaying blood were exploited to construct two acetylator congenic lines, A.B6 and B6.A, as explained in Figure 9.2 and the previous section. The availability of quartets shown in Figure 9.3 provided the means to test for the effects of background genes on NAT activity for various substrates and on the role of acetylation polymorphism on individual susceptibility to arylamine carcinogenesis. The background effect on NAT activity with AF, for example, indicated that the A background contributed a factor that increased the phenotypic difference in NAT activity with, for example, AF or conversely that the B6 background reduced the phenotypic difference in NAT activity.

Acetylator congenic lines were used to evaluate the role of acetylation polymorphism on individual susceptibility to arylamine carcinogenesis as measured over a short term (3 hours) by AF-induced hepatic DNA-adduct formation. Differences in the acetylator phenotype caused differences in the formation of DNA-AF adducts; for hepatic DNA, adduct formation in B6 mice was about twice that of A mice. The comparisons of parental to congenic mice were also revealing because the A.B6 mice had about 4-fold (for females) and 10-fold (for males) more hepatic DNA-AF adducts than A mice. The difference on the B6 background was somewhat less: B6 mice had 2.7-fold (females) and 1.4-fold (males) more hepatic DNA adducts than B6.A mice. These experiments indicated that differences in acetylator phenotype do contribute to differences in AF-induced DNA damage, and show that mouse gender can influence tissue-specific arylamine-induced DNA damage even though no gender-related differences in hepatic NAT activity have been found.

Another (subchronic 28 day) study of arylamine exposure and acetylator phe-notype was also performed using B6 and B6.A mice exposed to 4-aminobiphenyl, another arylamine with carcinogenic potential. Little or no relationship was observed between acetylator phenotype and liver damage, but in the urinary bladder, one of the major targets for arylamine carcinogens, rapid acetylator females had more adducts than slow acetylator females. However, slow acetylator males had higher bladder adducts than rapid acetylator males. Both the 28-day study with 4-aminobiphenyl and the short-term (3-hour exposure) tests with AF show higher adduct formation in liver for females compared to males and higher adduct formation in bladder for males compared to females. The relationship of sex to organ site of arylamine carcinogenesis indicated by both 3-hour and 28-day exposures is the same as has been observed in lifetime feeding studies. This observation suggests that measurement of DNA-carcinogen adducts may be an acceptable adjunct to lifetime tumor production in assessing carcinogenicity.

Another example of the use of the A.B6 and B6.A acetylator congenic mice in toxicology focused on the study of cleft palate (CP) and cleft lip with and without cleft palate [CL(P)].29 A mice are more susceptible to teratogen-induced and spontaneous CP than B6 mice. Using conventional genetic analysis of a number of AXB and BXA recombinant inbred lines, a few genetic markers including Nat on chromosome 8 were found for increased sensitivity to the teratogens phe-nytoin, glucocorticoids, and 6-aminonicotinamide. Refinement of these experiments through the use of acetylator congenic mice showed that the sensitivity to glucocorticoid-induced cleft palate was high in A and B6.A mice whereas B6 and A.B6 mice were resistant to the teratogenic effects. While Nat is considered unlikely to be the cause of the teratogenesis, the results indicate that the gene responsible for teratogen sensitivity is quite near Nat on mouse chromosome 8. Given the high degree of homology existing between the Nat-containing region of mouse chromosome 8 and human chromosome 8, the NAT* genes may also be useful markers for the study of CP and [CP(P)] in humans.

While these toxicological studies with congenic mice were in progress, recombinant DNA studies of Nat were advancing in another direction. Molecular biological techniques had shown that several mammalian species including humans are characterized by at least two genes that encode two very similar NAT proteins expressed in liver and other tissues. For the mouse, recombinant and heterologous expression studies revealed several points of interest. First, three Nat genes Natl*, Nat2*, and Nat3* are present, one of which (Nat2*) is polymorphic. The coding region sequences of Natl * for A and B6 mice are identical, while that for Nat2* from the slow acetylator A mice contains a single base missense mutation that is accompanied by replacement of asparagine by iso-leucine at position 99 in the coding region. Second, recombinant studies provide an explanation for the genetically variant patterns of N-acetylation of PABA, AF, and isoniazid. The Nat* genes transiently expressed in COS-l cells showed that Natl has selectivity for acetylation of isoniazid, Nat2 has absolute specificity for PABA, and Natl and Nat2 have overlapping specificities for AF. Mutant Nat2 from A mice with the N99I amino acid substitution is less active than its counterpart in B6 mice in the acetylation of PABA and AF, but the rates of isoniazid acetylation with Natl from A and B6 mice are comparable.

Molecular genetic and biochemical studies of B6 and A mice and congenic acetylator lines have provided insight into the mechanism for genetic slow acetylation. The evidence indicates that defective acetylation in slow acetylator mice is caused by a conformationally modified Nat2 enzyme (Nat2*9) with markedly reduced stability and with decreased affinity for the substrate.30 The missense mutation has no effect on the amount of Nat2 transcript or protein and the mouse mutant Nat2*9, unlike mutant Nat2 in human liver or in mammalian expression systems, is not subject to degradation by hepatic proteases. These mouse studies suggest that proteolytic processing of structurally altered proteins is not a universal phenomenon but more likely the type and extent of confor-mational modification introduced may dictate whether proteolysis takes place.

Further investigations of congenic mice and double congenic mice have revealed interactions between the polymorphisms of the Ah receptor and acet-yltransferases that affect the extent of DNA damage induced by exposure to arylamine carcinogens.3l,;32 A critical step in hepatic metabolic activation of arylamines appears to be N-oxidation catalyzed by the cytochrome P450, CYPlA2. This isozyme has been associated with arylamine metabolism in both mice and humans. In mice, CYP1A2 is a member of the gene battery controlled by the Ahr receptor locus, and hence differences in inducibility of such a key P450 enzyme might lead to differences in susceptibility to arylamine-induced cancer. The likelihood that both acetylation polymorphism and Ah receptor polymorphism could play an interactive role in the early stages of arylamine carcino-genesis led to the construction of the double congenic line, B6.A.D, a mouse line that is both a slow acetylator and a nonresponder. The quartet of lines on the B6 background, B6, B6.A, B6.D, and B6.A.D, was studied to examine the effect of induction of the Ah receptor by p-naphthoflavone on arylamine-induced hepatic DNA damage (Figure 9.5). The results were in agreement with previous observations on noninduced mice: rapid acetylators had more hepatic DNA-AF adducts than males, and females had a greater adduct burden than males. The greater adduct burden in livers of females of all strains was also found after induction, although since male mice responded to induction to a greater extent than did females, the differences in adduct formation between males and females after p-naphthoflavone treatment was reduced in lines that had a significant response to induction. This study demonstrated differences in arylamine-induced hepatic DNA damage in mice of differing acetylator status and Ahr responsiveness. Since all four lines tested shared the B6 genetic background, differences in DNA damage that were found can be attributed only to differences in responsiveness to the inducer and/or to differences in acetylation capacity. Other differences between the mice in this study arise from gender-related effects, and therefore results for males and females are considered separately.

A similar type of study using the same four lines of congenic and double congenic mice was performed to examine the effects of both polymorphisms on the effects arising from exposure to food mutagens (heterocyclic arylamines) derived from cooked meats and suspected of being cancer initiators.32 The results of these studies are complex, but in sum they confirm that Ahr and Nat genotypes and coexposure to an Ah agonist (such as p-naphthoflavone) contribute significantly in a tissue-specific way to the amount and profile of DNA-amine adducts formed in several organs, which in humans are target organs for the food mutagens.

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