Reproducibility studies and interlaboratory concordance for androgen assays in female plasma.

We conducted studies to determine the magnitude and sources of variability in androgen assay results and to identify laboratories capable of performing such assays for large epidemiological studies. We studied androstanediol (ADIOL), androstanediol glucuronide (ADIOL G), androstenedione (ADION), androsterone glucuronide (ANDRO G), androsterone sulfate (ANDRO S), dehydroepiandrosterone (DHEA), dehydroepiandrosterone sulfate (DHEA S), dihydrotestosterone (DHT), and testosterone (TESTO). A single sample of plasma was obtained from five postmenopausal women, five premenopausal women in the midfollicular phase of the menstrual cycle, and five women in the midluteal phase, divided into aliquots, and stored at -70 degrees. Four sets of two coded aliquots from each woman were then sent to participating labs for analysis at monthly intervals over 4 months. Using the logarithm of assay measurements, we estimated the components of variance and three measures of reproducibility. The usual coefficient of variation is a function of the components that are under the control of the laboratory. The intraclass correlation between measurements for a given individual is the proportion of the total variability that is associated with individuals. The minimum detectable relative difference is important to evaluate study feasibility. Results suggest that a single sample of ADIOL G, DHEA, DHEA S, and ANDRO G (with two lab replicates per sample) can be used to discriminate reliably among women in a given menstrual phase or menopausal status. The results for DHT, TESTO, ADION, and ANDRO S are more problematic and suggest that the present measurement techniques should be used with care, especially with midluteal phase women. The results for ADIOL suggest that this assay is not yet ready for use in epidemiological studies.

Reproducibility and validity of radioimmunoassays for urinary hormones and metabolites in pre- and postmenopausal women.

The reproducibility of RIAs of circulating sex hormones has been evaluated as part of recent epidemiological investigations, but none seem to have addressed the reproducibility or validity of RIAs for urinary hormones or their metabolites. As part of a case-control study of breast cancer in Asian-American women, 12-h overnight urine samples were obtained, and a methodological study was conducted to identify laboratories capable of assaying urinary hormones. For the reproducibility component of this study, two laboratories with extensive experience in hormone assays measured urinary estrone, estradiol, estriol, pregnanediol glucuronide, and estrone glucuronide using samples from 15 women (5 midfollicular, 5 midluteal, and 5 postmenopausal). Variance estimates from these measurements were used to calculate the laboratory variability (coefficient of variation) and to assess the magnitude of the biological variability among the women in relation to the total variability (intraclass correlation coefficient). For the validity component, urinary estrone, estradiol, and estriol levels were measured in the same samples by gas chromatography-mass spectroscopy in the laboratory of Dr. Herman Adlercreutz (University of Helsinki, Helsinki, Finland). We found that the degree of assay reproducibility differed between the laboratories, but that laboratory variability was usually low compared with the range of hormone values among women, particularly for the estrogens. Values for estrone and estradiol were well correlated among all of the laboratories. For estriol, the RIAs tended to overestimate levels compared with gas chromatography-mass spectroscopy. In one laboratory, assays for pregnanediol glucuronide and estrone glucuronide were consistently reproduced; in the other, the reproducibility of the RIA for pregnanediol glucuronide was problematic, and estrone glucuronide was not measured. Despite some limitations, urinary hormones and their metabolites can be reliably measured by current RIAs in large investigations attempting to link hormone level to disease risk and may be particularly advantageous for studies of postmenopausal women, where serum concentrations of estrone and estradiol are low and assay measurements are not as dependable.

Quantifying estrogen metabolism: an evaluation of the reproducibility and validity of enzyme immunoassays for 2-hydroxyestrone and 16alpha-hydroxyestrone in urine.

Rapid and simple enzyme immunoassays (EIAs) were recently developed to measure 2-hydroxyestrone and 16alpha-hydroxyestrone in unextracted urine. The balance between these competing estrogen metabolism pathways may serve as a biomarker of breast cancer risk. Before testing these assays in epidemiologic studies, we evaluated their reproducibility, and validity relative to gas chromatography-mass spectroscopy (GC-MS). Overnight 12-hr urine collections from five midfollicular premenopausal women, five midluteal premenopausal women, and five postmenopausal women were aliquoted and stored at -70 degrees C. Two aliquots from each woman were assayed with the EIAs in a random, blinded order, monthly over 4 months and 1 year later. Reproducibility over 4 months was good for both metabolites in premenopausal women (coefficient of variation = 8-14%) and satisfactory in postmenopausal women (approximately 19%). Reproducibility over 12 months remained good in premenopausal women, but was poor in postmenopausal women, with mean readings increasing 50 to 100%. Wide variation in estrogen metabolite levels enabled a single EIA measurement to characterize individual differences among premenopausal women in midfollicular (intraclass correlation coefficient = 98-99%) and midluteal phase (85-91%). A narrower range in metabolite levels among postmenopausal women reduced discrimination (78-82%). The correlation between EIA and GC-MS measurement was excellent for both metabolites (r>0.9), except for 2-hydroxyestrone in postmenopausal women (r=0.6). Analysis of absolute agreement suggested that both EIAs were less sensitive than GC-MS, and each detected nonspecific background. The low concentration of estrogen metabolites in urine from postmenopausal women may explain the problems with reproducibility and validity in this menstrual group. Accordingly, more sensitive EIAs have been developed and are now being evaluated.

Reproducibility studies and interlaboratory concordance for assays of serum hormone levels: estrone, estradiol, estrone sulfate, and progesterone.

We conducted studies to measure sources of assay variability for estrone, estradiol, estrone sulfate, and progesterone for postmenopausal women (n = 5) and for women in the mid-follicular (n = 5) and mid-luteal (n = 5) phases of the menstrual cycle. A single blood sample from each woman was divided into 2.5-ml aliquots and stored at -70 degrees C, and sets of two aliquots were sent at monthly intervals to each of three laboratories (four for progesterone). Each aliquot was analyzed in duplicate. Thus, within each menstrual category, we were able to estimate the components of variance due to variation among women, variation among aliquots, variation among duplicate measurements, and variation among the 4 analysis days. Using the logarithm of assay measurements, we estimated the percentage of variance attributable to variation among women in each menstrual category, 100 rho, is the estimated intraclass correlation. For each assay, 100 rho exceeded 90% for mid-follicular and mid-luteal women. For postmenopausal women, values of 100 rho exceed 84% for estrone in two laboratories. Values of 100 rho were lower for progesterone in postmenopausal women, although a value of 84% was estimated from one laboratory. These studies indicate that estrogen assays over a period of 3 months permit reliable comparisons among women in a given menstrual category. Progesterone measurements are likewise reliable for women in the mid-follicular and mid-luteal phases but somewhat less satisfactory for postmenopausal women. These assessments of variability pertain only to laboratory techniques and do not allow for secular variation in intra-woman hormone levels. Moreover, although these measurements tend to be reliable enough for making comparisons among women, estimates of coefficients of variation for estrogens are about 10% for mid-follicular and mid-luteal phase women and about 11-20% for postmenopausal women. Coefficients of variation for progesterone are about 10% for mid-luteal, 20% for mid-follicular, and 30% for postmenopausal women.

Increased risk of colorectal cancer among smokers: results of a 26-year follow-up of US veterans and a review.

To clarify the relationship between tobacco use and risk of colorectal cancer, we evaluated a cohort of 248,046 American veterans followed prospectively for 26 years. In comparison with veterans who had never used tobacco, the risk of death was significantly increased for colon cancer and rectal cancer among current and former cigarette smokers and among pipe or cigar smokers, controlling for social class and occupational physical activity. Rectal-cancer risk was also significantly elevated among users of chewing tobacco or snuff. For both sites, risk increased significantly with pack-years, earlier age at first use, and number of cigarettes. These results reinforce 2 recent reports of the association of cigarette smoking and colorectal cancer in men and women. Inconsistencies in the findings of earlier epidemiologic studies appear to be due in large part to differences in length of follow-up or in choice of controls. Studies with at least 20 years of follow-up or population-based controls have tended to find elevated risk with tobacco smoking, while those with shorter follow-up or hospital controls have not. This, plus the strength and consistency of the association of smoking and colon polyps, suggest that smoking may primarily affect an early stage in the development of colon cancer. If this association is causal, tobacco use may be responsible for 16% of colon-cancer and 22% of rectal-cancer deaths among these veterans.

The role of agricultural pesticide use in the development of non-Hodgkin's lymphoma in women.

Non-Hodgkin's lymphoma has been found to be associated with agricultural pesticide use in men, but little is known about the risk in women. In a recent population-based, case-control study conducted in eastern Nebraska, no increased risk of non-Hodgkin's lymphoma was found in women who had ever lived or worked on a farm (odds ratio [OR] = 1.0). Neither the use of insecticides (OR = 0.8) nor herbicides (OR = 0.7) on the farm was associated with non-Hodgkin's lymphoma; however, the number of women who mixed or applied pesticides was small, particularly in comparison to men on farms. Small nonsignificant associations were observed among the women who personally handled insecticides (OR = 1.3) or herbicides (OR = 1.2). Women who personally handled organophosphate insecticides had a significant 4.5-fold increased risk of non-Hodgkin's lymphoma. Use of chlorinated hydrocarbon insecticides was associated with an OR of 1.6; however, the use on dairy cattle was associated with a 3-fold increased risk. Pesticide-related risks were greater among women with a family history of cancer, particularly a history of lymphatic or hematopoietic cancer among first-degree relatives.

Soft tissue sarcoma and tobacco use: data from a prospective cohort study of United States veterans.

A report of an increased risk of soft tissue sarcoma (STS) among users of smokeless tobacco led us to evaluate this association and the role of other types of tobacco in a prospective cohort mortality-study of United States veterans. A total of 248,046 veterans provided tobacco-use histories on a mail questionnaire in 1954 or 1957. Data on subsequent tobacco use were not collected. By 1980, 119 deaths from STS had occurred among the cohort members. Veterans who had ever chewed tobacco or used snuff had a nonsignificant 40 percent excess of STS (95 percent confidence interval [CI] = 0.8-2.6; 21 deaths) in comparison with veterans who had never used any tobacco products. Risk was limited to former users (relative risk [RR] = 1.5) with no excess seen among current users (RR = 0.9). Frequent former users had higher risk (RR = 1.9) than infrequent users (RR = 1.3). Risk was slightly higher in persons who started using smokeless tobacco at younger ages, but did not increase with duration of use or with late age at cessation of use. Most veterans who used chewing tobacco or snuff also used some other form of tobacco. No STS deaths occurred among the 2,308 veterans who used smokeless tobacco only. An unexpected finding of the study was the significant excess of STS deaths among cigarette smokers (RR = 1.8, CI = 1.1-2.9). Risk was higher among ex-smokers (RR = 2.2) than among current smokers (RR = 1.5) and was not related to number of cigarettes per day, age started smoking, duration, or pack-years.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of hair coloring products and the risk of lymphoma, multiple myeloma, and chronic lymphocytic leukemia.

OBJECTIVES: Hair coloring products are widely used and contain components that are mutagenic and carcinogenic. An association between occupational exposure to hair coloring products and hematopoietic cancers has been reported, but the risk for these cancers among users has not been carefully evaluated. METHODS: We conducted a population-based, case-control study with telephone interviews from 385 with telephone interviews from 385 non-Hodgkin's lymphoma cases, 70 Hodgkin's disease cases, 72 multiple myeloma cases, 56 chronic lymphocytic leukemia cases, and 1432 controls. RESULTS: Among women, use was associated with odds ratios of 1.5 for non-Hodgkin's lymphoma, 1.7 for Hodgkin's disease, 1.8 for multiple myeloma, and 1.0 for chronic lymphocytic leukemia. Risk was higher for permanent hair coloring products than for semi- or nonpermanent products, particularly for dark colors. Long duration and early age of first use tended to increase risk, but the patterns were inconsistent. Use was much less common in men and did not significantly increase risk. CONCLUSIONS: The use of hair coloring products appears to increase the risk of non-Hodgkin's lymphoma. Multiple myeloma and Hodgkin's disease were also associated, although based on far fewer subjects. If these results represent a causal association, use of hair coloring products would account for 35% of non-Hodgkin's lymphoma cases in exposed women and 20% in all women.

A prospective study of tobacco use and multiple myeloma: evidence against an association.

The relationship between the use of cigarettes and other tobacco products and the risk of multiple myeloma was examined in a cohort of nearly 250,000 American veterans followed prospectively for 26 years. Compared with men who had never tobacco, the risk of death from myeloma was not increased among current (relative risk [RR] = 0.9, 95 percent confidence interval [CI] = 0.8-1.2) or former (RR = 1.0, CI = 0.8-1.3) cigarette smokers, nor among users of chewing tobacco or snuff (RR = 1.0, CI = 0.4-2.3). Risk was only slightly and nonsignificantly increased among pipe or cigar smokers (RR = 1.2, CI = 0.9-1.5). There was no indication of increasing risk with amount of tobacco used or earlier age at first use. With over 90 percent power to detect a 30 percent increased risk of this tumor occurring among current cigarette smokers, this study provides the strongest evidence to date against an association of cigarette smoking with multiple myeloma.

A case-control study of non-Hodgkin's lymphoma and the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in eastern Nebraska.

To evaluate the role of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in the development of non-Hodgkin's lymphoma (NHL), we conducted a population-based, case-control study in 66 counties in eastern Nebraska. Telephone interviews were conducted with 201 white men diagnosed with NHL between July 1, 1983, and June 30, 1986, and with 725 controls. There was a 50% excess of NHL among men who mixed or applied 2,4-D (odds ratio [OR] = 1.5; 95% confidence interval = 0.9, 2.5). The risk of NHL increased with the average frequency of use to over threefold for those exposed 20 or more days per year (p for trend = 0.051). Adjusting for use of organophosphate insecticides lowered the risk estimate for frequent users (OR = 1.8), but adjustment for fungicide use increased the risk estimate (OR = 4.5). Simultaneous adjustment for organophosphates and fungicides yielded an OR of 3.1 for farmers who mixed or applied 2,4-D more than 20 days per year. Risk also increased with degree of exposure, as indicated by application method and time spent in contaminated clothing, but not with the number of years of 2,4-D use or failure to use protective equipment. Although other pesticides, especially organophosphate insecticides, may be related to NHL, the risk associated with 2,4-D does not appear to be explained completely by these other exposures.

Metabolism of N-hydroxy-2-acetylaminofluorene and N-hydroxy-phenacetin by guinea pig liver microsomal enzymes.

Deacylation has been proposed as a mechanism of activation of arylhydroxamic acids. In the present studies solubilized preparations from guinea pig liver microsomes, a source of high deacylase activity, were subjected to gel filtration on Sephacryl S-200. A single peak (peak I) of activity was found when column fractions were assayed colorimetrically for deacylation of N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Corresponding to this peak were the following activities: binding of [3H-ring]-N-hydroxy-phenacetin (N-OH-P) to tRNA and deacylation of N-OH-P and N-OH-AAF, measured by the formation of nitrosophenetole (N = O-P) and nitrosofluorene (N = O-F), respectively. The binding of [3H-ring]-N-OH-AAF to tRNA was catalyzed by peak I, but to a greater extent by a second peak (II). The binding of both N-OH-P and N-OH-AAF to tRNA was inhibited by paraoxon, an esterase inhibitor. H.p.l.c. analysis revealed that for peak I, the major ether-extractable metabolites of N-OH-P and N-OH-AAF were the corresponding nitroso derivatives. In the presence of peak II, little metabolism to organic-extractable metabolites occurred. These data indicate that more than one mechanism is involved in the activation of N-OH-P and N-OH-AAF in this system, and that the difference in the activation of these arylhydroxamic acids cannot be explained by differences in the formation of deacylated metabolites.

Prostaglandin H synthase-mediated reaction of carcinogenic arylamines with tRNA and homopolyribonucleotides.

Prostaglandin H synthase mediates the reaction of an extensive series of carcinogenic arylamines with tRNA. Structure-activity relationships suggest that benzidine is especially reactive due to extended conjugation between the 4,4'-diamino groups. In trapping experiments with homopolyribonucleotides, benzidine reacts with polyguanylic acid but 4-aminobiphenyl reacts with polycytidylic acid. The nitrenium ion of 4-aminobiphenyl (formed by N,O-acyltransferase activation of N-hydroxy-4-acetylaminobiphenyl) reacts primarily with polyguanylic acid and to a lesser extent with polyadenylic acid. The results suggest that arylamine activation by prostaglandin H synthase does not involve nitrenium ion formation.

Population distribution of placental benzo(a)pyrene metabolism in smokers.

Human placental microsomes isolated from term placentas derived from nonsmoking women and women smoking 1 to 40 cigarettes a day were analyzed for the metabolism of benzo(a)pyrene measured as various metabolites by HPLC and/or as aryl hydrocarbon hydroxylase (AHH)6 activity. In accordance with other reports, AHH activity was several times higher in smokers than in nonsmokers. Regression analysis on 13 different placental tissues from women smoking from 1 to 40 cigarettes demonstrated a high correlation (r = 0.8 to 0.9) between AHH activity (or the formation of benzo(a)pyrene phenols resolved by HPLC) versus the formation of the procarcinogenic benzo(a)pyrene-7,8-diol. Subsequent studies on placentas derived from 67 women who smoked 10 to 40 cigarettes per day demonstrated a definite dose-response relationship between AHH activity and the number of cigarettes smoked/day. The dose-response curve was sigmoidal in shape; however, when the data were plotted on a semi-log scale the curve assumed a linear shape, reaching saturation of AHH induction beyond 20 to 25 cigarettes/day. While mean AHH activity was dependent upon the number of cigarettes smoked/day, considerable interindividual variability in AHH (ranging more than 1,000-fold in some cases) was observed among individuals with comparable smoking histories, i.e. smoking the same number of cigarettes. Population distribution suggested clustering of the population in the low-AHH-activity region while cord-blood thiocyanate analysis and twin studies suggested that genetic factors contributed to a major portion of the inter-individual variability in AHH activity observed among smokers.

Relationship of metabolic activation of N-hydroxy-N-acylarylamines to biological response in the liver and mammary gland of the female CD rat.

Intraperitoneal injection of the N-formyl, N-acetyl, or N-propionyl derivatives of N-hydroxy-4-aminobiphenyl, N-hydroxy-2-acetylaminofluorene, or N-(4-biphenyl)glycolamide disclosed that the ability of these compounds to induce mammary tumors in the female CD rat was greater if the compound was able to be metabolized to a reactive product by one of two soluble enzymes obtained from both the liver and mammary gland. A similar but weaker association between the formation of gamma-glutamyltranspeptidase-positive foci and cellular altered foci of the liver was also observed. The enzyme related to the tumorigenicity of these compounds was characterized by a highly specific capacity to form adducts from the acetyl and propionyl derivatives. The other enzyme exhibited greater activity with N-formyl substrates. The two enzyme activities were separable by ion-exchange chromatography on DEAE-cellulose and by gel filtration on Sephacryl. Liver microsomes also possessed the capacity to activate both the formyl and acetyl derivatives to reactive species; formyl substrates were 7 to 8 times more active than acetylated compounds. The microsomal activities and the formyl-preferring soluble enzyme were inhibited by diethyl-p-nitrophenylphosphate, a microsomal deacylase inhibitor. The cytosolic enzymes that are most active with the acetyl and propionyl substrates were little affected by this organophosphate compound. The microsomal activation was not due solely to deacylation of the hydroxamic acid, since formylated and acetylated substrates were hydrolyzed at approximately the same rates.

Activation of N-hydroxyphenacetin to mutagenic and nucleic acid-binding metabolites by acyltransfer, deacylation, and sulfate conjugation.

N-Hydroxyphenacetin was activated to a mutagen in the Salmonella-Ames test by rabbit liver acyltransferase, rat liver cytosol, and rat liver microsomes. N-[ring]3H]-Hydroxyphenacetin was bound to transfer RNA when activated by acyltransferase from rabbit or rat liver or rat liver microsomes. The acyltransferase-catalyzed binding was not inhibited by paraoxon, a deacetylase inhibitor. The use of N-hydroxyphenacetin radioactively labeled in the acetyl group, as well as the ring, indicated that deacetylation was involved in the microsome-catalyzed binding reaction. In addition, the microsome-catalyzed binding was inhibited 90% by paraoxon. p-Nitrosophenetole, a deacetylated derivative of N-hydroxyphenacetin, was synthesized and bound to transfer RNA without enzymatic activation. Activation of N-hydroxyphenacetin by sulfate conjugation was also found to lead to binding to transfer RNA. The data implicated acyl transfer, deacetylation, and sulfate conjugation as possible routes for the activation of N-hydroxyphenacetin.

Arylhydroxylamine-induced ribonucleic acid chain cleavage and chromatographic analysis of arylamine-ribonucleic acid adducts.

Reaction of N-hydroxy-2-aminofluorene (N-OH-AF) with rRNA at pH 5.0 decreased the molecular weight of the polynucleotide. Toluene-soluble aryl derivatives were released on hydrolysis of fluorenylamine- and biphenylamine-substituted RNA by treatment with venom phosphodiesterase and alkaline phosphatase. These data suggested that arylhydroxylamines, activated by incubation at pH 5.0 or by enzymatic O-acetylation, might react with the phosphate group of RNA to give unstable phosphate triesters. Spontaneous hydrolysis of these triesters would result in cleavage of the polynucleotide chain. Further enzymatic hydrolysis of the phosphate esters would yield nonpolar arylamine derivatives. Enzymatically degraded 4-aminobiphenyl(ABP)-RNA adducts were examined by high performance liquid chromatography (HPLC) for the presence of a putative phosphorylated adduct. Synthetic standards of the C-8-guanosine monophosphate-ABP adduct (ABP-GMP) and o-aminobiphenyl-O-phosphate were used as markers in the analysis of the digested RNA. A phosphate adduct of ABP was undetectable by these methods. The data also indicated that the ABP-GMP formed in the acyltransferase-mediated binding of N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) to RNA is readily degraded during the enzymatic digestion of the RNA adduct.

Benzo[a]pyrene metabolism and macromolecular binding in strains of Ah responsive and Ah non-response mice.

Using liver microsomes as the enzyme source in in vitro assays, benzo[a]pyrene (BP) metabolism was studied in eight inbred strains of mice (C57BL/6J, DBA/2HaD, BALB/cCR, AKR/Sn, RF/J, CBA/J, C57L/J and 129/J). BP metabolite formation was monitored by high pressure liquid chromatography (HPLC) and by following aryl hydrocarbon hydroxylase (AHH) activity. Other parameters measured in cluded the formation of alkali-extractable radioactivity due to [3H] BP metabolites, microsomal protein binding, DNA binding and epoxide hydrase activity. The induction of BP metabolism and binding to macromolecules was studied after treatment of animals with phenobarbital (PB) or 3-methylcholanthrene (MC). Four of the mouse strains (C57BL/6J, BALB/cCr, CBA/J and C57L/J) were highly inducible with respect to liver AHH when pretreated with MC. The induction of AHH by MC in these strains correlated well with the radioactive metabolites of [3H] BP remaining in the alkali extract derived from the AHH assay mixture and with the increased binding of [3H] BP to microsomal protein and DNA. In addition to the eight strains listed above, eight recombinant inbred lines showed a positive correlation between AHH induction and induction of DNA-binding metabolites. PB pretreatment resulted in less than two-fold induction of AHH and alkali-extractable radioactivity. However, DNA and microsomal protein binding were induced by PB pretreatment more than AHH. Ratios of MC-induced/basal activity for BP-phenols were very similar to induction ratios of AHH activity determined by the fluorometric method. BP-quinone formation was induced to the same extent as phenols. This relationship did not hold for dihydrodiol formation; dihydrodiol induction was often higher than AHH or phenol induction. For MC-pretreated mice, dihydrodiol induction, as determined by HPLC, did not parallel macromolecular binding induction as closely as did AHH. For PB-pretreated mice, dihydrodiol induction was as poor as indicator of binding induction as AHH. Epoxide hydrase activity, using styrene oxide as substrate, was induced markedly by PB-pretreatment, but very little by MC-pretreatment. Epoxide hydrase induction did not parallel BP-dihydrodiol induction when microsome preparations from MC- or PB-treated mice were used. These data suggest this enzyme is not rate limiting in this system.

Evidence that arylhydroxamic acid N,O-acyltransferase and the genetically polymorphic N-acetyltransferase are properties of the same enzyme in rabbit liver.

Arylhydroxamic acid N,O-acyltransferase and Co-ASAc-dependent N-acetyltransferase activities were measured simultaneously in liver cytosols from rabbits of known acetylator phenotype. Both activities were high in rapid acetylator rabbits and low in slow acetylator rabbits indicating that these two acetyl transfer steps in the metabolic activation of certain arylamines are under common genetic control in this species. The two enzyme activities could not be resolved by sequential centrifugation, fractional precipitation with ammonium sulfate, ion exchange chromatograhy on DEAE-cellulose, gel filtration on Sephacryl, and electrophoresis on polyacrylamide gels. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis of extracts of disc gel electrophoretic slices showed that a single symmetrical protein band with a molecular weight of 33,000 was associated with both activities. The results obtained strongly suggest that the CoASAc-dependent N-acetyltransferase reaction and the intramolecular N,O-acetyl transfer by arylhydroxamic acid N,O-acyltransferase are properties of the same enzyme.

High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism by human lymphocytes from donors of different aryl hydrocarbon hydroxylase inducibility and antipyrine half-lives.

With high-pressure liquid chromatography (HPLC), lymphocytes from six human donors were evaluated for their ability to metabolize benzo(a)pyrene (BP). Donors whose aryl hydrocarbon hydroxylase (AHH) inducibility ratios ranged from 2.4 to 4.6 and whose antipyrine plasma half-lives ranged from 8 to 17 hr were examined. The BP metabolites identified were: 7,8-dihydrodiol, quinones, and 9-hydroxy and 3-hydroxy phenols. HPLC profiles of BP metabolites elaborated by uninduced (control) and benz(a)anthracene-induced lymphocytes were qualitatively similar among the six donors. A good correlation (r = 0.79) was found between known AHH inducibility ratios for the donors, as determined by the conventional fluorometric AHH assay, and induction of BP phenol production quantitated from HPLC data. HPLC results also indicated that the induction of benzo(a)pyrene-7,8-dihydrodiol, the proposed proximate carcinogenic form of BP, did not parallel BP phenol induction. Furthermore, the data also indicated a good negative correlation between AHH inducibility and the measurements of plasma antipyrine or urinary 4-hydroxyantipyrine half-lives (r = -0.88 or -0.91), respectively.