Duodenal carcinoma in MUTYH-associated polyposis.

Bi-allelic germline mutations in the MUTYH gene give rise to multiple adenomas and an increased incidence of colorectal cancer. In addition, duodenal adenomas and other extra-colonic manifestations have been described in MUTYH-associated polyposis (MAP) patients. We describe two patients with bi-allelic MUTYH gene mutations with duodenal carcinoma. The tumour in Patient A was detected during evaluation of non-specific abdominal complaints. Patient B was already diagnosed with tens of adenomas and a colon carcinoma, when a duodenal neoplasm was detected. The identification of somatic G>T mutations in codon 12 of the K-RAS2 gene provides evidence that the duodenal lesions were induced by MUTYH deficiency. Studies in larger series of MAP patients are needed to investigate the risk of upper-gastro-intestinal malignancies and to determine further guidelines for endoscopical surveillance.

Computer-based diagnostic support systems in histopathology: what should they do?

In many fields in histopathology diagnosis making is notoriously difficult. We explored the diagnostic process in the area of CL and related disorders. The field of cutaneous lymphomas (CL) and borderline lesions is complex and representative for the type of diagnostic problems encountered. A review of diagnostic support systems in diagnostic pathology revealed that the usefulness of these systems has been disappointing. We contribute this to the fact that these systems target only part of the diagnostic process. A tentative model of diagnosis making in pathology is presented. This model assumes a two-step process, from observation to feature recognition and from features to diagnosis. In a retrospective study of existing skin biopsy pathology reports we assessed detail and scope of the histological descriptions. In a second, prospective study, a pathologist panel described a set of 16 skin biopsies using a standard set of descriptors. The retrospective study showed a large variability in the nature and details of described features, whereas the prospective study showed lack of consensus regarding both feature descriptions and diagnostic category. Both studies provide an indication that lack of consensus in feature recognition may be an important contributor to lack of consensus at the diagnostic level. Diagnostic expert systems target the step from feature to diagnosis. Evidently different input into such systems produces different output. We conclude that support of the feature recognition step can contribute to better diagnostic consensus because of more uniform interpretation of observations.

Familial cancer database: a clinical aide-mémoire.

Cancer is associated with a wide range of hereditary disorders. Recognizing these disorders in cancer patients may be of great importance for the medical management of both patients and their relatives. Conversely, recognizing the fact that cancer may develop in patients already diagnosed with a particular hereditary disorder may be important for the same reason. We have developed a stand-alone interactive computer program, Familial Cancer Database (FaCD), to assist the clinician in making a genetic differential diagnosis in cancer patients as well as in becoming aware of the tumour spectrum associated with a particular hereditary disorder. The program tries to match tumour and non-tumour features observed in patients and their families with any of the more than 300 disorders presently contained in its database and provides a clinical synopsis with literature references for each of these disorders. FaCD is offered free of charge in support of the Familial Cancer and Prevention project of the UICC Cancer Epidemiology and Prevention program. The software is primarily aimed at health care professionals with at least a basic knowledge of clinical cancer genetics, and can be downloaded from the internet at http://facd.uicc.org.

CA 125 in serum and tumor from patients with uterine sarcoma.

Serial serum samples of 33 patients with primary sarcoma of the uterus were analyzed for CA 125 and frozen tissue sections of tumor from 23 patients were tested for this antigen. Before treatment, 12 of 30 evaluable patients showed serum CA 125 levels> 16 Uml-1 (40%). There was no relationship between serum CA 125 level and the histologic subtype. Patients with serum CA 125> 16 Uml-1 showed extrauterine tumor sites in 67% of the cases versus 33% in patients with normal CA 125 determinations (P = 0.026). In (FIGO) stages I and II, elevated serum CA 125 levels prior to surgery were associated with a poor prognosis (P = 0.043). Patients with recurrent or progressive disease demonstrated serum CA 125 levels> 16 Uml-1 in 14 of the 20 cases (70%). Sarcoma cells were completely negative for CA 125, whereas positivity was observed in the epithelial component of mixed Müllerian tumors. The source of the elevated serum CA 125 levels in patients with uterine sarcoma may be stimulated mesothelial cells.

Interspecies variations in the histology of toxicologically important areas in the larynges of CRL:CD rats and Syrian golden hamsters.

Specific regions in the rodent larynx exhibit cellular changes in response to inhaled xenobiotics. These regions include the base of the epiglottis, ventral pouch, and medial surfaces of the vocal processes of the arytenoid cartilages. There are interspecies differences among laboratory rodents in the microscopic anatomy of these sensitive areas of the laryngeal mucosa. In CRL:CD strain Sprague-Dawley rats, the mucosa covering the epiglottis differs from that of Syrian golden hamsters. The epithelium covering the base of the epiglottis is relatively thin in rats and is composed of a mixture of cell types, whereas in hamsters it is much thicker and is made up almost entirely of tall ciliated columnar cells. The cartilage supporting the ventral pouch in the larynges of hamsters is much more prominent than in rats and forms a distinct protrusion into the laryngeal lumen at the base of the epiglottis. The purpose of this paper is to describe and illustrate these and other subtle differences in rat and hamster laryngeal anatomy, which may be of toxicologic significance.

Bioassay for carcinogenicity of rotenone in female Wistar rats.

Rotenone, a pesticide extracted from the Derris root, consistently was reported by a series of investigators to have induced mammary fibroadenomas in female Wistar rats when administered ip or by gavage in a sunflower (SF) oil or SF oil:chloroform vehicle. In contrast, no less than eight bioassays done in other laboratories with rotenone or rotenone-containing powders have given consistently negative carcinogenic results when different strains or species and different modes or vehicles of administration have been used. However, these studies were not designed to address the biological reproducibility of the positive data. Thus, the present study was designed to simulate conditions of the positive studies and to investigate a possible cocarcinogenic interaction between rotenone and chloroform. Each of eight treatment groups was assigned 72 weanling female Wistar rats. Groups were (1) untreated, (2) needle puncture, (3) SF oil:10% chloroform (SF oil:chloroform), (4) 1.0 mg/kg rotenone in SF oil:chloroform, (5) 2.0 mg/kg rotenone in SF oil:chloroform, (6) SF oil, (7) 1.0 mg/kg rotenone in SF oil, and (8) 2.0 mg/kg rotenone in SF oil. Rats were injected ip 5 days a week for 8 weeks (42 injection days) and subsequently held for 16 months. The appearance of palpable tissue masses was recorded; over 50 tissues from each rat were histologically evaluated. There were no statistically significant differences in overall or individual tumor incidences among control and rotenone-treated groups. Specifically, neither incidence nor time-to-palpation of mammary fibroadenoma significantly differed among control and rotenone-treated groups, regardless of the vehicle of administration. Thus, rotenone was not carcinogenic, and rotenone and chloroform did not interact to produce a carcinogenic effect in female Wistar rats in the current study. Thus, previous reports of carcinogenic activity were not reproducible under similar experimental conditions.

Histological sectioning of the rodent larynx for inhalation toxicity testing.

In rodents, the larynx is a major site of histopathologic alteration following inhalation exposure to particulates, vapors, and aerosols. Specifically, the epithelial lining of a narrowly delineated region on the ventral floor of the larynges of rats and mice appears to be especially vulnerable to inhaled materials, and is recognized as a preferred site for histopathological evaluation in inhalation studies. This site is located at the base of the epiglottis, cranial to the ventral laryngeal diverticulum (ventral pouch). The presence of underlying seromucinous glands is critical for histologic identification of this site. We report a histologic sectioning technique, using the ventral laryngeal diverticulum as the anatomical landmark, to obtain tissue sections from this area of predilection in rats and in mice.

Comparative genotoxicity testing of mainstream whole smoke from cigarettes which burn or heat tobacco.

The genotoxic potential of mainstream whole smoke (MWS) from cigarettes which heat tobacco (TEST) was compared to the genotoxic potential of MWS from a cigarette which burns tobacco (REFERENCE). MWS was collected from a University of Kentucky 1R4F cigarette (REFERENCE) and two, TEST cigarettes, one with regular flavor and the other with menthol flavor. All cigarettes were smoked on a smoking machine and the particulate phase was collected on Cambridge filter pads. The vapor phase, which passed through the pad, was bubbled into a dimethyl sulfoxide (DMSO) trap. The filter pad was extracted with the DMSO in the trap and additional DMSO to obtain MWS. MWS representing an identical number of cigarettes was tested to make a per-cigarette comparison of their genotoxic potential. REFERENCE MWS was mutagenic and cytotoxic in the Ames assay in the presence of metabolic activation while it was cytotoxic but not mutagenic in the absence of metabolic activation. Statistically significant increases in frequency of both sister-chromatid exchanges and chromosomal aberrations were observed in Chinese hamster ovary cells exposed to REFERENCE MWS with and without metabolic activation. MWS from the TEST cigarettes, with either regular or menthol flavor, was neither cytotoxic nor mutagenic in any of these assays. In summary, MWS from the 2 TEST cigarettes was neither genotoxic nor cytotoxic under conditions where MWS from the REFERENCE cigarettes was genotoxic and/or cytotoxic in a concentration-dependent manner.

Comparative studies of the mutagenicity of urine from smokers and non-smokers on a controlled non-mutagenic diet.

Measuring the mutagenicity of urine is widely viewed as a means of evaluating human exposure to potentially genotoxic materials. Diet and cigarette smoking have both been reported to affect the mutagenicity of human urine, but the relationship between smoking status and the expression of diet-related urinary mutagenicity is unknown. It has been reported that some promutagens are more active in in vitro assays when tested in the presence of urine from smokers than when tested in the presence of urine from non-smokers. We aimed to determine whether the differences in urinary mutagenicity between smokers and non-smokers result from increased urinary mutagenicity from dietary heterocyclic amine mutagens in smokers compared with non-smokers. Groups of smokers and non-smokers (6-12) were given identical diets, previously shown to be low in heterocyclic amines and very low in mutagenicity. The diet consisted exclusively of raw food and of food cooked in boiling water. After a 2-day dietary stabilization period, 24-hour urine samples were collected for three consecutive days. The regimen was repeated in the following week. For comparison, both groups were also placed on a "western" diet, consisting of a variety of foods prepared by several cooking methods, designed to reflect what a typical United States family might consume. Urine was concentrated using XAD-2 resin and then assayed for mutagenic activity in the Ames test. The urine of smokers was significantly more mutagenic than that of non-smokers when on both the raw/boiled and the "western" diets. These results indicate that the increased urinary mutagenicity observed in smokers compared with non-smokers is not due to enhanced mutagenicity of diet-related heterocyclic amine mutagens in the urine of smokers.

Chemical and biological studies of a cigarette that heats rather than burns tobacco.

Cigarettes can be developed that heat rather than burn tobacco. Such products would be expected to have less "tar" and other combustion products than cigarettes that burn tobacco. With one product of this type, benzo(a)pyrene, N-nitrosamines, phenolic compounds, acetaldehyde, acrolein, hydrogen cyanide, and N-heterocyclic compounds have been reduced 10- to 100-fold compared to the Kentucky reference (1R4F) cigarette, a representative low-tar cigarette. The yields of nicotine and carbon monoxide from this new cigarette are less than the yields of 95% and 75%, respectively, of the cigarettes sold in the United States during 1988. Nicotine absorption from smoking this new cigarette is not significantly different from that of tobacco-burning cigarettes yielding equivalent levels of nicotine. The urine mutagenicity of smokers of new cigarettes is significantly less (P less than .05) than that of smokers of tobacco-burning cigarettes and is not significantly different (P greater than .10) from that of nonsmokers. We conclude that cigarettes which heat rather than burn tobacco can reduce the yield of tobacco combustion products. This simplification of smoke chemistry had no effect on nicotine absorption in smokers and resulted in a reduction of biological activity in smokers as measured by urine mutagenicity.

Genetic toxicology studies comparing the activity of sidestream smoke from cigarettes which burn or only heat tobacco.

The results of in vitro genetic toxicology studies of sidestream cigarette smoke (SSCS) from cigarettes which heat but do not burn tobacco were compared to those of sidestream smoke from cigarettes which burn tobacco. SSCSs from 5 cigarettes were compared. Three of the cigarettes, the Kentucky reference research cigarette (1R4F), a commercially available ultra-low-tar brand (ULT) and a commercially available ultra-low-tar menthol brand (ULT-menthol) burn tobacco while two of the cigarettes, a regular (TEST) and a menthol (TEST-menthol) heat tobacco. SSCSs from all cigarettes were prepared by identical techniques, which involved collecting sidestream smoke particulate matter on Cambridge filter pads and combining the particulate matter with the vapor-phase materials collected by bubbling the smoke exiting the Cambridge pad through DMSO. The SSCSs obtained (equivalent to 0.4 cigarettes/ml DMSO) were evaluated at identical concentrations in an in vitro genetic toxicology test battery. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in Ames bacterial strains TA98, TA100, TA1537 and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative results in strain TA1535. In the absence of metabolic activation, 1R4F, ULT and ULT-menthol SSCSs were not significantly mutagenic. TEST and TEST-menthol SSCSs produced negative results in all 5 bacterial strains, both with and without metabolic activation. SSCS from 1R4F, ULT and ULT-menthol cigarettes produced positive results in the CHO chromosomal aberration assay and in the CHO sister-chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol SSCSs produced negative results in both assays, either with or without metabolic activation. The SSCSs from 1R4F, ULT and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol SSCSs were negative in this assay. All 5 SSCSs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. SSCSs from the 1R4F, ULT and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol SSCSs were not cytotoxic under either condition. These results demonstrate that sidestream smoke from cigarettes which heat but do not burn tobacco (TEST and TEST-menthol) was neither genotoxic nor cytotoxic under conditions where sidestream smoke from cigarettes which burn tobacco (1R4F, ULT and ULT-menthol) was genotoxic and/or cytotoxic in a concentration-dependent manner.

Comparative studies on the genotoxic activity of mainstream smoke condensate from cigarettes which burn or only heat tobacco.

The in vitro genotoxic activity of mainstream cigarette smoke condensate (CSC) from cigarettes which heat but do not burn tobacco was compared to that of CSC from cigarettes which burn tobacco. CSCs from five cigarettes were compared. Three of the cigarettes [the Kentucky reference research cigarette (1R4F), a commercially available ultra-low tar brand (ULT) and a commercially available ultra-low tar menthol brand (ULT-menthol]) burn tobacco while two of the cigarettes [a regular (TEST) and a menthol (TEST-menthol]) heat tobacco. CSC from all cigarettes were collected by identical standard techniques, which involved collecting mainstream smoke particulate matter on Cambridge filter pads under FTC smoking conditions. The pads were extracted with DMSO, and the CSCs obtained [10 mg total particulate matter (TPM)/ml DMSO] were evaluated at identical concentrations in an in vitro genetic toxicology test battery. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were mutagenic in Ames bacterial strains TA98, TA100, TA1537, and TA1538 in the presence of metabolic activation (S9 from Aroclor-induced rat liver) but negative in strain TA1535. In the absence of metabolic activation, 1R4F, ULT, and ULT-menthol CSCs were not mutagenic except for a weak response in strain TA1537 for the 1R4F and ULT CSCs. TEST and TEST-menthol CSCs were nonmutagenic in all five bacterial strains, both with and without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were positive in the CHO-chromosomal aberration assay and in the CHO--sister chromatid exchange assay both with and without metabolic activation while TEST and TEST-menthol CSCs were negative in both assays, either with or without metabolic activation. CSCs from 1R4F, ULT, and ULT-menthol cigarettes were weakly positive in inducing DNA repair in cultured rat hepatocytes while TEST and TEST-menthol CSCs were negative in this assay. All five CSCs were nonmutagenic in the CHO-HGPRT assay both with and without metabolic activation. CSCs from the 1R4F, ULT, and ULT-menthol cigarettes were cytotoxic in the CHO-HGPRT assay, both with and without metabolic activation, while TEST and TEST-menthol CSCs were not cytotoxic under either condition. These results demonstrate that mainstream CSCs from the TEST and TEST-menthol cigarettes are neither genotoxic nor cytotoxic under conditions where CSCs from 1R4F, ULT, and ULT-menthol cigarettes are genotoxic and/or cytotoxic in a concentration-dependent manner.

Histologic changes in the respiratory tract induced by inhalation of xenobiotics: physiologic adaptation or toxicity?

Toxicologists and pathologists are often faced with the dilemma of categorizing changes observed in the respiratory tract of laboratory animals as either "adaptive" or "toxic." However, it is often difficult to interpret the nature of a given change as either "adaptive" or "toxic." Certain lesions or changes in the respiratory tract are to be expected from the concentration of materials given or the experimental design of a study. Careful analysis suggests that some of these changes may be more properly described as adaptive rather than toxic within the context of a given study or situation. Tissue changes discussed in this paper include squamous metaplasia of laryngeal epithelium, goblet cell change in respiratory epithelium, macrophage accumulation within alveoli, and bronchiolization of alveolar epithelium. Examples provided show that some of these changes observed in inhalation studies are similar in severity but slightly increased in frequency over sham control animals. The introduction of exogenous material into the respiratory tract of laboratory animals in an experimental setting should be expected to result in certain changes. The challenge scientists must accept is to interpret these changes so that toxic events may be separated from adaptive changes. In order to meet this challenge, studies incorporating several species and novel technologies may have to be utilized.

Effect of cooking methods on the mutagenicity of food and on urinary mutagenicity of human consumers.

The effects of cooking methods on the in vitro mutagenicity of individual foods, the in vitro mutagenicity of meals containing those foods, and the mutagenic exposure of human volunteers following consumption of the meals were examined using Ames bacterial strain TA98 with S-9 metabolic activation. Three methods of food preparation--boiling, baking and frying/flame-broiling--were compared. With meats, frying or broiling resulted in higher in vitro mutagenicity (10- to 50-fold) than did baking or boiling, whereas for carbohydrates, eggs or vegetables mutagenicity did not vary markedly with cooking method. The observed (experimental) mutagenic activity of the meals was quite similar to their calculated (predicted) mutagenicity, obtained by summing the mutagenicity of the individual foods in the meal. The close agreement between experimental and predicted mutagenicity indicated that components of the meal did not interact in either a synergistic or inhibitory manner. The mutagenicity of fried flame-broiled meals was approximately 10-fold greater than the mutagenicity of baked or broiled meals, which were similar in mutagenicity. The mutagenicity of human urine following consumption of the meals was related to the in vitro mutagenicity of the meals themselves. The in vitro mutagenicity of meals is markedly affected by the cooking method used to prepare them and the mutagenicity of the diet may be reflected in the mutagenicity of body fluids.

Ninety-day inhalation study in rats, comparing smoke from cigarettes that heat tobacco with those that burn tobacco.

Eight groups of 30 male and 30 female rats were exposed 1 hr per day, 5 days per week for 13 weeks, to smoke from reference (tobacco burned) or test (tobacco only heated) cigarettes, at nicotine concentrations of 5, 15, or 30 micrograms/liter of air. Similar smoke concentrations of wet total particulate matter and carbon monoxide were produced in each of the test/reference comparisons. There was a pronounced depression of minute ventilation of animals in the reference groups, but not in the test animals. Blood carboxyhemoglobin concentrations were similar in animals exposed to smoke from test and reference cigarettes. Plasma concentrations of nicotine and cotinine in the test groups were higher than in the reference groups. There were no differences between the smoke-exposed groups in terms of body weight or feed consumption. At necropsy, an increase in heart weight was noted in both high exposure groups. There were notable differences in histopathology, with fewer and less-pronounced changes in the test groups than in the reference groups. Many of the histopathological responses induced in the reference groups were absent in the test groups. Overall, the study demonstrated a substantial reduction in the biological activity of smoke from the test cigarette when compared with the reference.

Human urine mutagenicity study comparing cigarettes which burn or only heat tobacco.

Cigarette smokers have been reported to void urine which is more mutagenic, as measured in the Ames bacterial mutation assay, than urine voided by non-smokers. Condensate from the mainstream smoke of a cigarette which heats, but does not burn tobacco (test cigarette) showed no evidence of mutagenicity in a battery of in vitro genotoxicity assays under conditions in which condensate from the mainstream smoke of cigarettes that burn tobacco was mutagenic. The objective of this study was to determine whether the absence of mutagenic activity observed in the in vitro assays would be reflected in the urine of smokers of the test cigarette. 72 subjects (31 smokers and 41 non-smokers) were enrolled in a 6-week study, with the smokers randomly divided into 2 groups. The study was designed as a double crossover, with each smoker smoking both test (tobacco-heating) and reference (tobacco-burning) cigarettes. This design allowed each smoker to serve as his or her own control while at the same time allowing comparisons between groups of non-smokers and smokers of both test and reference cigarettes. 24-h urine samples were collected twice a week and concentrated using XAD-2 resin. Urine concentrates were tested in Ames bacterial strains TA98 and TA100, with and without metabolic activation and with and without beta-glucuronidase/aryl sulfatase. Individuals who smoked the test cigarette voided urine which was significantly less mutagenic than that voided when they smoked reference cigarettes. The mutagenicity of urine from smokers who smoked the test cigarette and non-smokers did not differ under any of the assay conditions used in this study.

Absorption of nicotine from a cigarette that does not burn tobacco.

Ten smokers participated in a study to compare the absorption of nicotine from the smoke aerosol of a new cigarette that heats, but does not burn tobacco (test) with a cigarette that burns tobacco (reference). The average plasma nicotine concentrations obtained by the 7th test cigarette (13 ng/ml) and 7th reference cigarette (24 ng/ml) were proportional to the nicotine yielded by the two cigarettes as determined under Federal Trade Commission machine-smoking conditions. These data demonstrate that the smoke aerosol obtained by smoking a cigarette which heats tobacco produces plasma profiles of nicotine that are similar to the profiles obtained from smoking a cigarette that burns tobacco.

Carcinogenesis of 4-aminobiphenyl in BALB/cStCrlfC3Hf/Nctr mice.

Male and female (840 each) BALB/cStCrlfC3Hf/Nctr mice were given 0, 7, 14, 28, 55, 110 and 220, and 0, 7, 19, 38, 75, 150 and 300 ppm, respectively, of 4-aminobiphenyl in their drinking water. Necropsies on killed animals were performed at 13, 26, 39, 52 and 96 weeks on dose. Dose-related neoplasms were angiosarcomas, bladder urothelial carcinomas and hepatocellular neoplasms. The non-neoplastic dose-related lesions were left atrial thrombosis, bladder urothelial hyperplasia, splenic hemosiderosis and splenic erythropoiesis. The incidences of bladder carcinoma and atrial thrombosis were higher in the males and the incidences of hepatocellular neoplasms and angiosarcomas were higher in the females.

Rat mammary gland carcinogenesis after local injection of N-hydroxy-N-acyl-2-aminofluorenes: relationship to metabolic activation.

A single local injection of 2.5 mumol of N-hydroxy-N-formyl-2-aminofluorene (N-hydroxy-FAF), N-hydroxy-N-acetyl-2-aminofluorene (N-hydroxy-AAF), or N-hydroxy-N-pro-pionyl-2-aminofluorene )N-hydroxy-PAF) to each of the six left mammary glands of female Sprague-Dawley derived CD rats gave a mammary tumor incidence, after 12 months, of 53% for the N-acetyl (42% adenocarcinoma, 11% fibroadenoma), 41% for the N-formyl (8% adenocarcinoma, 11% sarcoma, 22% fibroadenoma), and 33% for the N-propionyl (11% adenocarcinoma, 22% fibroadenoma) derivatives of N-hydroxy-N-2-aminofluorene, Latent periods for malignant tumor appearance (adenocarcinoma or sarcoma) was 210 days, 148 days, and 177 days, respectively, with no malignant tumors occurring in the vehicle-treated animals. In contrast, latent periods for benign tumor appearance (fibroadenoma) was 263 days for control animals, 289 days for the N-hydroxy-AAF, 324 days for the N-hydroxy-FAF, and 317 days for the N-hydroxy-PAF animals. When N-acetyl-2-aminofluorene (AAF) was applied as above there was only an 8% mammary tumor incidence (4% adenocarcinoma, 4% fibroadenoma) with a latent period of 207 days for malignant tumor (adenocarcinoma) and 221 days for benign tumor (fibroadenoma) appearance. Arylhydroxamic acid N, O-acyltransferase activity has been demonstrated in the mammary glands of male, and lactating and non-lactating female Sprague-Dawley derived CD rats by means of nucleic acid binding assay. Mammary gland cytosol catalyzed tRNA adduct formation to a greater extent with N-hydroxy-FAF. AAF was not activated by this enzyme. Ammonium sulfate fractionation demonstrated the presence of two enzymes, one specific for N-hydroxy-FAF (70-80% fraction), the other specific for N-hydroxy-AAF and N-hydroxy-PAF (40-70% fraction). Moreover, gel filtration chromatography of mammary gland cytosol demonstrated the presence of two enzymes of differing acyl specificity. Mammary gland microsomes catalyzed the formation of tRNA adducts, but only with the N-hydroxy-FAF derivative. Assays that tested the mutagenic potential of the arylhydroxamic acids in Salmonella typhimurium TA-1538 with either mammary gland cytosol or microsomes demonstrated the order of mutagenicity to be N-hydroxy-FAF greater than N-hydroxy-AAF greater than N-hydroxy-PAF. A similar order of mutagenicity was demonstrated without an external metabolic activation system. These data demonstrate that the presence of two distinct enzymes in the rat mammary gland that activate arylhydroxamic acids.