Purification of hepatic polymorphic arylamine N-acetyltransferase from homozygous rapid acetylator inbred hamster: identity with polymorphic N-hydroxyarylamine-O-acetyltransferase.

The polymorphic acetyltransferase isozyme expressed in homozygous rapid acetylator inbred hamster liver cytosol was purified over 2000-fold by sequential Q-Sepharose fast-flow anion-exchange chromatography, Sephacryl S-200 high-resolution size-exclusion chromatography, Mono Q anion-exchange fast-protein liquid chromatography, and preparative polyacrylamide gel electrophoresis. The isozyme migrated as a single homogeneous monomer following both preparative and sodium dodecyl sulfate-polyacrylamide electrophoresis. The molecular weight was estimated at 34,170 following elution via size-exclusion chromatography and 35,467 following migration via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The homogeneous polymorphic acetyltransferase exhibited a broad substrate specificity; it catalyzed the acetyl coenzyme A-dependent N-acetylation of p-aminobenzoic acid, carbocyclic arylamine carcinogens such as 2-aminofluorene, 4-aminobiphenyl and beta-naphthylamine, and heterocyclic arylamine carcinogens such as 2-aminodipyrido[1,2-a:3'2'd]imidazole and 3-amino-1-methyl-5H-pyrido[4,3-b]indole. It also readily catalyzed the acetyl coenzyme A-dependent metabolic activation (via O-acetylation) of N-hydroxy-2-aminofluorene to DNA adducts but not the metabolic activation (via intramolecular, N,O-acetyltransfer) of N-hydroxy-2-acetylaminofluorene or N-hydroxy-4-acetylaminobiphenyl to DNA adducts. Conversely, the partially purified monomorphic acetyltransferase isozyme from the same hamsters readily catalyzed the metabolic activation of N-hydroxy-2-acetylaminofluorene and N-hydroxy-4-acetylaminobiphenyl, and rates of metabolic activation of these substrates did not differ between homozygous rapid and slow acetylator liver, intestine, kidney, and lung cytosols. Heat inactivation rates for the purified polymorphic acetyltransferase isozyme were first order and indistinguishable for the acetyl coenzyme A-dependent N-acetylation and O-acetylation activities. The results strongly suggest the expression of a single polymorphic acetyltransferase product of the hamster polymorphic acetyltransferase gene that catalyzes both acetyl coenzyme A-dependent N-acetylation and O-acetylation of arylamine and N-hydroxyarylamine carcinogens but not the metabolic activation of N-hydroxy-N-acetylarylamines (arylhydroxamic acids) via intramolecular N,O-acetyltransfer. Consequently, acetylator genotype-dependent metabolic activation of N-hydroxyarylamines to a DNA adduct in hamster is catalyzed by direct O-acetylation of the hydroxyl group and not via sequential N-acetylation followed by N,O-acetyltransfer.

Corneal endothelial dystrophy. A study of 64 families.

A prospective study was undertaken during an 18-month period with 64 families who had endothelial dystrophy. Two hundred twenty-eight relatives were examined. Of those older than the age of 40, 38% were affected. Women were affected more severely and 2.5 times more frequently than men. The disease showed a strong familial tendency: there was one family in which three generations were affected and 16 families in which two generations were affected. There were four families that had members with edema in two generations. There was no association between edema in a parent and edema in a child. The proportion of relatives affected and the severity of involvement increased with age. Fifty-three probands and 18 relatives had endothelial dystrophy with edema (Fuchs' dystrophy). Of these 71, one had glaucoma.

Transmission of affective disorders: an application of segregation analysis to blind family study data.

The single major locus (SML) model of Bucher and Elston (1981) was applied to data collected in a long-term follow-up and family study of major effective disorders. Pedigree segregation analysis was used to test the hypothesis that bipolar and unipolar disorders are phenotypic variants of the same genotype at a SML. The particular SML model examined did not adequately describe the familial pattern of these disorders in families of bipolar and unipolar probands. Although other SML models may be superior, it is likely that the existence of a SML will be obscured by genetic heterogeneity.

Acetyltransferases and susceptibility to chemicals.

Arylamine chemicals inflict a number of toxicities including cancer. Metabolic activation (i.e., oxidation) is required in order to elicit the toxic actions. Acetylation is an important step in the metabolic activation and deactivation of arylamines. N-acetylation forms the amide derivative which is often nontoxic. However, O-acetylation of the N-hydroxyarylamine (following oxidation) yields an acetoxy arylamine derivative which breaks down spontaneously to a highly reactive arylnitrenium ion, the ultimate metabolite responsible for mutagenic and carcinogenic lesions. Human capacity to acetylate arylamine chemicals is subject to a genetic polymorphism. Individuals segregate into rapid, intermediate, or slow acetylator phenotypes by Mendelian inheritance regulated by a single gene encoding for a polymorphic acetyltransferase isozyme (NAT2). Individuals homozygous for mutant alleles are deficient in the polymorphic acetyltransferase and are slow acetylators. A second acetyltransferase isozyme (NAT1) is monomorphic and is not regulated by the acetylator genotype. Several human epidemiological studies suggest an association between slow acetylator phenotype and urinary bladder cancer. In contrast, a few studies suggest a relationship between rapid acetylator phenotype and colorectal cancer. The basis for this paradox may relate to the relative importance of N- versus O-acetylation in the etiology of these cancers. Conclusions drawn from human epidemiological data are often compromised by uncontrolled environmental and other genetic factors. Our laboratory recently completed construction of homozygous rapid, heterozygous intermediate, and homozygous slow acetylator congenic Syrian hamsters to be homologous in greater than 99.975% of their genomes. The availability of these acetylator congenic lines should eliminate genetic variability in virtually all aspects of arylamine carcinogenesis except at the acetylator gene locus. Ongoing studies in these congenic hamster lines should provide unequivocal information regarding the role of genetic acetylator phenotype in susceptibility to arylamine-related cancers.

Behavioral and genetic interrelationships between locomotor activity and brain biogenic amines.

1. Spontaneous locomotor activity of four mouse strains, the albino ICR, BALB/C, the black C57 BL/6 and the brown CDF-I, was studied in conjunction with whole brain content of select biogenic amines and major metabolites. The ICR and C57BL/6 mice scored the highest and lowest motility among the strains studied, respectively. 2. The ICR mice motility were the most sensitive to experimental stress among the mouse strains evaluated. 3. Brain dopamine concentrations were greater in ICR and CDF-I mice than the other two mouse strains. This is compared to high serotonin and 5-hydroxyindole acetic acid levels determined in C57BL/6. 4. A relationship between the ICR mouse strain with a high motility and the ratios of brain dopamine:homovanillic acid and between serotonin:5-hydroxyindole acetic acid was established. 5. An inverse relationship between ICR mice motility and the ratio of metanephrine and normetanephrine:3-methoxy-4-hydroxyphenylglycol (MHPG) was determined. 6. It is concluded that genetic predisposition may account for a relationship between motor activity and brain biogenic amines which may give insight into the susceptibility of different patients to the development of certain extrapyramidal diseases.

A search for 'schizophrenia spectrum disorders'. An application of a multiple threshold model to blind family study data.

Family data from schizophrenic and control probands were analyzed using a descriptive analysis and multiple threshold models to determine whether a given group of diagnoses made in accordance with ICD-9 was aetiologically related to schizophrenia. The proportion of relatives receiving any psychiatric diagnosis, other than schizophrenia and affective disorder, was essentially the same between the two study groups. Furthermore, the data did not fit the multiple threshold model tested. Thus, the hypothesis that schizophrenia and a spectrum of disorders defined according to ICD-9 have a common familial aetiology was not accepted.

Testing the monogenic theory of schizophrenia: An application of segregation analysis to blind family study data.

Segregation analysis was applied to blind family data concerning schizophrenia to decide if the transmission of schizophrenia could be explained by a single major gene. Our results showed that the Mendelian model was unacceptable. Therefore, the monogenic hypothesis could not account for the transmission of schizophrenia. Since the hypothesis of no parent-child transmission was also not accepted, there was an indication that some form of vertical transmission existed which could be psychosocial, or an interaction between genetic and psychosocial factors. Our results suggest genetic heterogeneity in schizophrenia. Currently available clinical criteria for defining subgroups must be improved in conjunction with detection of biological indicators so that segregation analysis of family data could be effectively used in determining modes of transmission in schizophrenia.

Inheritance of endothelial dystrophy of the cornea.

64 families containing a proband with corneal endothelial dystrophy were examined in order to study the hereditary nature of the disease. Data concerning the frequency of occurrence, severity of the disease, ratio of affected females to males, relationship of the disease with age, and other factors were the subject of a previous report. 7 pedigrees which reflect features of endothelial dystrophy within the 64 families are presented. These features include multiple females in a family being affected, multiple consecutively affected generations, the occurrence of offspring with disease more severe than the parent, and endothelial decompensation (edema) at a relatively young age (less than 40 years of age). The importance of examining family members whenever possible rather than relying on history alone is emphasized. A statistical analysis of the inheritance pattern was performed. Endothelial dystrophy does not seem to follow a strict autosomal dominant pattern even though superficial inspection suggests autosomal dominant inheritance (both males and females affected, successive generations affected, 38% of relatives over the age of 40 years affected). Even though we were unable to determine a specific genetic mode of inheritance in these 64 families with endothelial dystrophy, we do feel that endothelial dystrophy is at least in part an inherited disease. Future investigations might prove sex-linked dominance, genetic heterogeneity, the influence of environmental factors, or a multifactorial etiology.

Polymorphic and monomorphic expression of arylamine carcinogen N-acetyltransferase isozymes in tumor target organ cytosols of Syrian hamsters congenic at the polymorphic acetyltransferase locus.

A number of human epidemiological investigations suggest a relationship between acetylator phenotype and the incidence and/or severity of tumors caused by exposure to arylamine carcinogens. Conclusions drawn from these investigations can be compromised by a variety of environmental and other genetic factors. To eliminate variability in these other factors, our laboratory recently completed construction of homozygous rapid (Bio. 82.73/H-Patr), heterozygous intermediate (Bio. 82.73/H-Patr/Pat(s)) and homozygous slow (Bio. 82.73/H-Pat(s)) acetylator congenic hamsters. The purpose of the present study was to assess the utility of this congenic hamster model for investigations into the relationship between acetylator genotype and arylamine carcinogenesis. We report the expression of acetylator genotype-dependent (polymorphic) and acetylator genotype-independent (monomorphic) N-acetyltransferase isozymes in hepatic cytosols. The hepatic polymorphic N-acetyltransferase isozyme isolated from the congenic hamsters expressed clearly acetylator-genotype dependent (Patr greater than Patr/Pat(s) greater than Pat(s)) N-acetylation towards p-aminobenzoic acid, 4-aminobiphenyl, 2-aminofluorene, p-aminophenol, 1-aminopyrene, 5-aminosalicylic acid, beta-naphthylamine, 3,4-dichloroaniline, 3,2'-dimethyl-4-aminobiphenyl and p-phenetidine. Acetylator genotype-dependent N-acetylation for a number of arylamines also was observed in liver, colon, kidney and urinary bladder cytosols derived from the congenic hamster lines, including arylamines highly carcinogenic to hamster colon and urinary bladders. It is concluded that the congenic hamster model will be useful in studies to delineate the role of acetylator genotype in the incidence or severity of arylamine tumors.

The Muscatine Cholesterol Family Study: familial aggregation of blood lipids and relationship of lipid levels to age, sex and hormone use.

The Muscatine Cholesterol Family Study includes data on first, second and third-degree relatives of school children who have the following cholesterol levels: above the 95th percentile in two consecutive school screens, N = 53; below the 5th percentile twice, N = 45; and a random sample between the 5th and 95th percentiles twice, N = 44. For males and females not taking estrogen the relationship of cholesterol to age was the same in high, middle, and low groups, although the overall level differed. For females taking estrogen, cholesterol levels rose more quickly with age in the high group as compared to the middle and low groups. The triglyceride levels of females taking estrogen is higher at younger ages and shows no significant change with increasing age. Among first degree relatives, the cholesterol correlations are between 0.25 and 0.30. Correlations in relatives of the middle group probands are higher than those in relatives of high or low group probands, but not significantly so. Correction for ascertainment increases the correlations in the high and low groups. Among second degree relatives cholesterol correlations in all three groups are approximately 0.20. There is negligible cholesterol correlation between spouses in the high and low groups, while the middle group spouse correlation is significantly greater than zero. Categorical analysis suggests the effect of the proband's cholesterol level is seen most upon the cholesterol level of parents, with siblings and second-degree relatives being less similarly affected.

The Muscatine Cholesterol Family Study: distribution of cholesterol levels within families of probands with high, low and middle cholesterol levels.

The Muscatine Cholesterol Family Study includes the relatives of three groups of schoolchildren: those with cholesterol levels above the 95th percentiles in two consecutive school screens, those with cholesterol levels below the 5th percentile twice, and random sample of those with cholesterol levels between the 5th and 95th percentiles twice. This paper examines the cholesterol distribution within and among these families for evidence of major gene effects. Three screening techniques are used: admixture analysis using maximum likelihood, comparison of within-sibship variances among groups, and regression of within-sibships variance on within-sibship mean. The results of the three techniques are consistent and indicate the existence of a major gene in a subset of the families with a proband above the the 95th percentile, but not in the other two groups of families. We estimate that 15% of schoolchildren with a cholesterol level above the 95th percentile twice, or 3 per thousand in the general population, have a dominant gene as a cause of the cholesterol elevation.

A genetic study of panic disorder pedigrees.

Pedigree analysis is done on 19 kindreds of panic disorder, and the results suggest that this disorder is transmitted as an autosomal dominant trait. Seven of these 19 kindreds were ascertained through a panic disorder proband with mitral valve prolapse. When the analysis is done omitting these seven kindreds, the results also suggest that panic disorder without prolapse is transmitted as an autosomal dominant trait.

Extrahepatic expression of the N-acetylation polymorphism toward arylamine carcinogens in tumor target organs of an inbred rat model.

An N-acetylation polymorphism is described that is expressed toward arylamine carcinogens in tumor target organs of an inbred rat model. High levels (rapid acetylator phenotype) of arylamine carcinogen N-acetyltransferase activity were observed in kidney, colon, prostate and urinary bladder cytosols derived from Fischer (F-344) inbred rats, the strain most commonly used for tumor bioassay studies and the strain most particularly used in arylamine-induced colon and prostate cancer studies. Significantly lower (slow acetylator phenotype) levels of arylamine carcinogen N-acetyltransferase activity were observed in corresponding tissue cytosols derived from Wistar-Kyoto inbred rats. Intermediate levels of arylamine carcinogen N-acetyltransferase activity significantly different from both the parental strains were observed in F1 hybrids of the parental strains, consistent with codominant expression of two alleles at a single gene locus. The arylamine substrates exhibiting the acetylator phenotype-dependent N-acetyltransferase activities included p-aminobenzoic acid, p-aminosalicylic acid, p-phenetidine, p-aminophenol, 2-aminofluorene, 3,2'-dimethyl-4-aminobiphenyl, beta-naphthylamine and 4-aminobiphenyl, but not procainamide. Highest levels of arylamine carcinogen N-acetyltransferase were expressed consistently in colon cytosol, but expression of the N-acetylation polymorphism toward arylamine carcinogens was observed in each (kidney, colon, prostate and urinary bladder) of the tumor target organs. The expression of the N-acetylation polymorphism in tumor target organs suggests that the inbred rat model will be useful in assessing the role of acetylator phenotype in arylamine-induced cancers of the colon and prostate.

Segregation analysis of low levels of high-density lipoprotein cholesterol in the collaborative Lipid Research Clinics Program Family Study.

Complex segregation analysis with the unified mixed model in white families from nine lipid research clinics was carried out to delineate the mode of familial transmission of plasma high-density-lipoprotein cholesterol (HDL-C). Three groups of families from the collaborative Lipid Research Clinics Program Family Study were assessed: 1,146 selected at random, 483 obtained through hypercholesterolemic probands, and 177 selected from the random sample because a number had low HDL-C, the sample sizes being 4,279, 1,807 and 735, respectively. The data were first transformed and adjusted for effects of covariates. Analyses were performed within clinic and selection strata and also pooled across clinics within strata. The results were consistent across strata and identified two major HDL-C clusters with means separated by approximately 3 SD. There was significant evidence of transmission of a major factor for low HDL-C, but transmission did not conform to Mendelian segregation expectations. There was also evidence of significant multifactorial transmission. Since low HDL-C levels are a major independent risk factor for coronary heart disease, the association of a major factor with familial aggregation of low HDL-C emphasizes the importance of detailed within-family sampling for low HDL-C after identifying a proband whose predominant dyslipoproteinemia is low HDL-C.

Acetylator genotype-dependent N-acetylation of arylamines in vivo and in vitro by hepatic and extrahepatic organ cytosols of Syrian hamsters congenic at the polymorphic acetyltransferase locus.

Our laboratory recently reported the successful construction of homozygous rapid (Bio. 82.73/H-Patr) and homozygous slow (Bio. 82.73/H-Pat(s)) acetylator congenic Syrian hamsters. These hamsters are isogenic except for the polymorphic acetylator gene locus (Pat) and perhaps other closely linked loci. The purpose of the present investigation was to assess the expression of acetylator genotype both in vivo and in vitro in a variety of hepatic and extrahepatic organ cytosols. Levels of arylamine N-acetyl-transferase were generally high and in the relative order: liver greater than colon greater than kidney greater than pancreas greater than prostate, urinary bladder, and lung. However, an acetylator gene dose-response was clearly expressed in each tissue, with highest levels in homozygous Patr acetylators, intermediate levels in heterozygous Patr/Pat(s) acetylators, and lowest levels in homozygous Pat(s) acetylators. The magnitude of the acetylator genotype-dependent differences in N-acetyltransferase activity were substrate specific, wherein p-aminobenzoic acid showed the largest differences and p-aminophenol the smallest. The N-acetylation of p-aminobenzoic acid in vivo also reflected acetylator genotype in the congenic hamsters. These results further document the successful construction of rapid and slow acetylator congenic hamsters which should prove very valuable in future studies to assess the role of acetylator genotype in the toxicity and carcinogenicity of arylamine chemicals.

Acetylator phenotype-dependent and -independent expression of arylamine N-acetyltransferase isozymes in rapid and slow acetylator inbred rat liver.

Although mouse, hamster, and rabbit models of the human N-acetylation polymorphism have been identified and characterized, many investigations of arylamine toxicity and carcinogenicity are carried out in the rat, particularly the Fischer 344 (F-344) inbred rat. We partially characterized a new rat model of the N-acetylation polymorphism by determining expression of arylamine N-acetyltransferase activities in liver cytosols derived from adult male inbred F-344, WKY, and their F1 hybrid rat strains. Levels of N-acetyltransferase activity differed significantly between the strains for many arylamine substrates, with highest levels in F-344, lowest levels in WKY, and intermediate levels in F1 hybrids of these two parental strains. However, for some other arylamine substrates, levels of N-acetyltransferase activity did not differ significantly between the rat strains. Partial purification of rat liver cytosols from the three strains resulted in identification of two N-acetyltransferase isozymes. The levels of N-acetyltransferase activity of one isozyme differed significantly between strains analogous to the pattern observed in crude cytosol. In contrast, the levels of N-acetyltransferase activity of the second isozyme did not differ between the strains. Based upon these results, the F-344 inbred strain is designated a rapid acetylator phenotype, the WKY inbred strain is designated a slow acetylator phenotype, and F1 hybrids of the two parental strains are designated intermediate acetylator phenotype. The identification of acetylator phenotype-dependent and -independent hepatic N-acetyltransferase isozymes in the inbred rat mimics the biochemical basis for acetylator phenotype-dependent and -independent expressions of N-acetylation in humans and other mammalian species.

Biological and cultural sources of familial resemblance in plasma lipids: a comparison between North America and Israel--the Lipid Research Clinics Program.

Heterogeneity in determinants of familial resemblance of lipid and lipoprotein levels between populations in North America and Israel was investigated using path analysis. A common protocol, identical measurement techniques, and the same statistical procedures were used in the two samples. Both genetic (h2) and cultural (c2) determinants of inheritance were significant for all lipid variables in the two studies. Genetic and cultural heritability of total cholesterol (h2 = 0.61, c2 = 0.02), low-density lipoprotein cholesterol (h2 = 0.59, c2 = 0.02), and high-density lipoprotein cholesterol (h2 = 0.55, c2 = 0.06) did not differ significantly between North America and Israel, while there was a significant difference for triglyceride (h2 = 0.41, c2 = 0.07 in North America; h2 = 0.61, c2 = 0.05 in Israel). Secondary parameters of the path model describing intrafamilial environmental relationships differed between the two countries. In particular, there was a higher correlation between marital environments in Israel for all traits except triglyceride, and a larger effect of father's environment on offspring's environment in Israel for all traits. Within both populations, variation of plasma lipids and lipoproteins was mostly explained by genetic factors and random unmeasured environmental factors. The contribution of common family environment was found to be small, though statistically significant. This is probably due to homogeneity of the distribution of familial environmental determinants within both countries.

Parent-offspring aggregation of plasma lipids in selected populations in North America and Israel. The Lipid Research Clinics Prevalence Study.

Parent-offspring associations of total cholesterol and triglycerides were compared between family dyads in six North American populations examined between 1972 and 1976 as part of the North American Lipid Research Clinics Prevalence Study and those from families examined between 1976 and 1979 at the Lipid Research Clinic located in Jerusalem. Common study design, protocol, and laboratory techniques were used by all Lipid Research Clinics. The authors first examined homogeneity of familial correlations across clinics in the North American population and across origin groups in the Israeli sample. In general, correlations were homogeneous across clinics and origin groups, except for parent-daughter pairs for triglycerides in North America. The pooled familial correlations were similar in the two study populations. There was no asymmetry in parent-offspring correlations by the sex of the offspring. The pooled mother-child correlations were significantly higher than father-child values in the North American sample only. The strength of parent-offspring similarity showed no consistent pattern of change with level of education of parents in either study group. Patterns of familial similarity are discussed in relation to genetic, cultural, and environmental differences between the two study populations.