Multicenter study to assess potential hazards from exposure to lipid peroxidation products in soya bean oil from Trilucent breast implants.

In response to a Hazard Notice by the Medical Devices Agency of the UK in 2000 regarding the Trilucent breast implant (TBI), an expert panel was convened to implement a research program to determine whether genotoxic compounds were formed in the soybean oil filler (SOF) of TBIs and whether these could be released to produce local or systemic genotoxicity. The panel established a research program involving six laboratories. The program recruited 47 patients who had received TBIs (9 patients had received silicone implants previously). A reference group (REBI) of 34 patients who had exchanged either silicone (17 patients) implants (REBI-E) or patients (17) who were to receive primary implantation augmentation with silicone (REBI-PIA), and who were included as needed to increase either the pre- or post-explantation sample number. Of the 17 REBI-E patients, 5 had silicone implants and 12 had saline implants previously (prior to the last exchange). Investigation was undertaken before and after replacement surgery in the TBI patients and before and after replacement or augmentation surgery in the REBI patients. The pre- to post-operative sample interval was 8-12 weeks. Pre-operative samples were collected within 7 days prior to the operation. Information on a variety of demographic and behavioral features was collected. Biochemical and biological endpoints relating to genotoxic lipid peroxidation (LPO) products potentially formed in the SOF, and released locally or distributed systemically, were measured. The SOF of explanted TBIs was found to have substantial levels of LPO products, particularly malondialdehyde (MDA), and low levels of trans-4-hydroxy-2-nonenal (HNE) not found in unused implants. Mutagenicity of the SOF was related to the levels of MDA. Capsules that formed around TBIs were microscopically similar to those of reference implants, but MDA-DNA adducts were observed in capsular macrophages and fibroblasts of only TBI capsules. These cell types are not progenitors of breast carcinoma (BCa) and the location of the implants precludes LPO products reaching the mammary epithelial cells which are progenitors of BCa. Blood levels of LPO products were not increased in TBI patients compared to REBI patients and did not change with explantation. In TBI patients, white blood cells did not show evidence of increased levels of LPO-related aldehyde DNA adducts. In conclusion, based on a number of measured parameters, there was no evident effect that would contribute to breast or systemic cancer risk in the TBI patients, and the recommended treatment of TBI patients involving explantation was judged appropriate.

Chicken egg fetal liver DNA and histopathologic effects of structurally diverse carcinogens and non-carcinogens.

Chicken egg fetal livers were evaluated for histopathological changes produced by four genotoxic hepatocarcinogens: 2-acetylaminofluorene (AAF), aflatoxin B1 (AFB1), benzo[a]pyrene (BaP), diethylnitrosamine (DEN); four structurally related non- or weakly- carcinogenic comparators: fluorene (FLU), aflatoxin B2 (AFB2), benzo[e]pyrene (BeP), N-nitrosodiethanolamine (NDELA); two epigenetic hepatocarcinogens: clofibric acid (CFA), phenobarbital (PB); and the non-carcinogen, D-mannitol (MAN). CFA, PB and MAN were also assessed for formation of DNA adducts using the 32P nucleotide postlabeling (NPL) assay and for DNA breaks using the comet assay. CFA was also assessed in enhanced comet assay for oxidative DNA damage induction. Eggs were dosed on days 9- 11 of incubation. For genotoxicity evaluation, livers were collected 3h after the last dose. Liver qualitative histopathology assessment was performed on days 12 and 18 of incubation. CFA was negative for DNA adducts but yielded clear evidence of DNA breaks due to oxidative stress. PB and MAN produced no DNA adducts or breaks. Liver to body weight ratios were not affected in most groups, but were decreased in DEN groups, and increased after PB dosing. Livers from control groups, FLU, AFB2, BeP, NDELA, CFA, and MAN groups, displayed a typical hepatocellular trabecular pattern at both time points. In contrast, the four genotoxic carcinogens induced time- and dose- related interference with fetal liver cell processes of proliferation, migration and differentiation, leading to hepatocellular and cholangiocellular pleomorphic dysplasia and re-(de-) differentiation with distortion of the trabecular pattern. In addition, dosing with the high dose of DEN produced gallbladder agenesis. PB induced hepatocellular hypertrophy, interference with migration, expressed as distortion of the trabecular pattern, and a moderate cholangiocellular dysplasia. In summary, histopathological analysis of chicken fetal livers revealed developmental anomalies, as well as genotoxicity-induced and, in the case of PB, adaptive morphological changes. Thus, the model provides histopathological outcomes of molecular effects.

Assessment of chronic toxicity and carcinogenicity in an accelerated cancer bioassay in rats of Nifurtimox, an antitrypanosomiasis drug.

The chronic toxicity and carcinogenicity of Nifurtimox (NFX), a 5-nitrofuran derivative used in the treatment of American trypanosomiasis, were studied in male and female Wistar rats in an accelerated cancer bioassay (ACB). The ACB is a mechanistic initiation/promotion chronic toxicity and carcinogenicity bioassay designed to assess potential carcinogenic activity of a test substance in critical organs and tissues of rodents in which human carcinogens are active. The organs studied were liver, lungs, urinary bladder (UB), mammary gland (MG), bone marrow, spleen, kidneys, colon, stomach and any grossly observed lesions. NFX is a genotoxin which has been reported previously to exert a variable degree of carcinogenic activity in rat liver, kidney, UB and MG. The present study was undertaken to assess whether NFX has initiating activity in these four named target sites. In the initiation phase, groups of 20 Wistar rats were given NFX daily in the diet at 0.2% for the first 12 weeks of the study to assess initiating activity, followed by promoters (PROs) for four organs for an additional 24 weeks. NFX was compared to the following known initiators (INs) for each of these four tissues: diethylnitrosamine (DEN) for liver and kidney, N-butyl-N(4-hydroxybutyl)nitrosamine (BBN) for UB and 7,12-dimethylbenz(a)anthracene (DMBA) for MG. PROs included phenobarbital (PB) for liver and kidney, nitrilotriacetic acid (NTA) for UB, and diethylstilbestrol (DES) for MG. NFX was also administered continuously without PROs for 40 weeks. At the end of dosing (40 weeks) and at the end of recovery (52 weeks), animals were sacrificed and subjected to complete gross and histopathological examinations, along with evaluations of body weight gain over time and terminal body weights. Mortality was highest with DEN+PB (group 6) (40%), followed by BBN+NTA (group 7) (15%) and NFX+DES (group 5) and DMBA+DES (group 8) (10% each). The same groups also showed significant reductions in body weight gain over time and terminal body weights at sacrifice. In these groups, the expected preneoplastic, neoplastic and metastatic neoplastic lesions were produced, demonstrating the sensitivity of the model. In groups given NFX+PROs (groups 3-5), either no neoplasms occurred (group 4) or only single neoplasms (groups 3 and 5). In contrast, the PROs all elicited tumors in groups given INs (groups 6-8). Also, NFX given alone for 40 weeks did not produce any chronic toxicity, preneoplastic or neoplastic lesions. Thus, in this study, NFX did not demonstrate chronic toxicity or carcinogenicity. Moreover, in four target sites, i.e., liver, kidney, UB and MG, it exhibited no neoplastic initiating activity manifested by PROs for these four target sites.

Major difference in the hepatocarcinogenicity and DNA adduct forming ability between toremifene and tamoxifen in female Crl:CD(BR) rats.

The hepatoproliferative effects of 2 antiestrogens, tamoxifen and toremifene, were compared in a sequential 15-month study in which 2 doses of each compound were administered by daily gavage to female Sprague-Dawley rats for up to 12 months. The doses were 11.3 and 22.6 mg/kg for tamoxifen and 12 and 24 mg/kg for toremifene. There were scheduled sacrifices at 3, 6, 12, and 15 months, the latter including a 3-month recovery period from the 12th through the 14th month. In the chronic toxicity study, tamoxifen at 22.6 mg/kg produced 100% incidence of hepatocellular carcinoma at the 12- and 15-month sacrifice intervals and 67% and 71% incidences at the 11.3-mg/kg dose. Sequential observations showed an increased incidence of glutathione S-transferase-positive foci of hepatocellular alteration by 3 months with tamoxifen in the absence of hepatotoxicity, with the first liver carcinoma appearing by 6 months of treatment. Unscheduled deaths occurring beyond 7.5 months in the tamoxifen treated groups were due in almost all cases to liver cancer. In striking contrast, toremifene did not produce any hepatoproliferative effects at 12- and 24-mg/kg dose levels, nor in a pilot study at 48 mg/kg. The 24-mg/kg dose of toremifene exerted an inhibiting effect on foci of hepatocellular alteration in rat liver detectable by glutathione S-transferase immunohistochemistry at 3 months and by conventional histology at 12 months. An antiproliferative effect was also evident in mammary gland and anterior pituitary where both toremifene and tamoxifen suppressed tumor incidence in comparison to the control group. The ability of these drugs to modify rat liver DNA after p.o. administration was investigated using the 32P-postlabeling assay. Administration of tamoxifen at 45 mg/kg for 7 days produced liver DNA nucleoside modifications represented by 7 spots on the autoradiogram. Unlike tamoxifen, toremifene did not produce any modified bases in rat liver DNA detectable by the 32P-postlabeling technique. The dose levels of tamoxifen that are strongly hepatocarcinogenic in the rat are compared with doses used in humans in various applications. Taking internal drug exposure into account, we conclude that the margin of safety for use of tamoxifen as an endocrine prophylactic agent for healthy, but breast cancer prone, women is questionable.

Inhibition of lung carcinogenesis by black tea in Fischer rats treated with a tobacco-specific carcinogen: caffeine as an important constituent.

Here, we examined the effect of black tea and caffeine on lung tumorigenesis in F344 rats induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in a 2-year bioassay. NNK was administered s.c. at a dose of 1.5 mg/kg body weight three times weekly for 20 weeks. Animals were given either black tea as drinking water at concentrations of 2%, 1%, or 0.5%, or caffeine in drinking water at concentrations identical to those in 2% and 0.5% tea infusions for 22 weeks. The treatment period began 1 week before and ended 1 week after the NNK administration. The animals were sacrificed on week 101 for the examination of tumors in target organs, including lung, liver, nasal cavity, and other major organs. The NNK-treated group, given 2% black tea, showed a significant reduction of the total lung tumor (adenomas, adenocarcinomas, and adenosquamous carcinomas) incidence from 47% to 19%, whereas the group given 1% and 0.5% black tea showed no change. The 2% tea also reduced liver tumor incidence induced by NNK from 34% in the group given only deionized water to 12%. The tumor incidence in the nasal cavity, however, was not affected by either black tea or caffeine at any of the concentrations tested. The most unexpected finding was the remarkable reduction of the lung tumor incidence, from 47% to 10%, in the group treated with 680 ppm caffeine, a concentration equivalent to that found in the 2% tea. This incidence is comparable to background levels seen in the control group. This study demonstrated for the first time in a 2-year lifetime bioassay that black tea protects against lung tumorigenesis in F344 rats, and this effect appears to be attributed, to a significant extent, to caffeine as an active ingredient of tea.

The urinary bladder carcinogen propoxur does not produce genotoxic effects in the urinary bladder of Wistar male rats.

Propoxur (PPX) is a carbamate insecticide which induced urinary bladder cancer in Wistar rats when fed at 5000ppm in Altromin 1321 diet (1321). In the present investigation, PPX was studied for induction of several key events related to modes of action (MOA) of carcinogenicity in urinary bladders (UBs). Wistar rats were administered the compound for 28 days at 8000ppm in Provini Liba SA 3883 diet, which is similar to the 1321 diet. o-Anisidine HCl (AH) was used as a genotoxic UB carcinogenic comparator, and trisodium nitrilotriacetate (NTA) as an epigenetic UB carcinogen comparator. Along with the non-dosed control and three test substance groups (PPX, AH, NTA), four more groups were additionally fed 2% ammonium chloride (AC) in the diet to acidify the urine, since 1321 was reported to increase urinary pH. AC did acidify the urine, as expected, although the 3883 diet itself did not increase pH values above 8. In the alkaline comet assay, AH produced DNA single strand breaks (SSBs) in the UB urothelium (UBU) irrespective of AC administration, whereas PPX and NTA did not. In the nucleotide (32)P-postlabeling assay (NPL), AH produced DNA adducts irrespective of AC administration, whereas PPX and NTA did not. Routine (H&E) histopathology evaluation of the UBU did not reveal any hyperplasia or evidence of luminal microprecipitates or calculi in any of the groups. Assessment of UBU proliferation as measured by immunohistochemistry of proliferating cell nuclear antigen, revealed that NTA and NTA plus AC increased the replicating fraction (RF). Also AH plus AC, but not AH alone, increased the RF of UBU, whereas PPX groups were not significantly different from controls. Thus, the results reveal no evidence for DNA SSBs, binding, or alteration of DNA synthesis in the UBU by PPX, while demonstrating UBU DNA damage by AH and showing that NTA does not damage DNA, but causes increased UBU proliferation. The findings are in accord with a genotoxic MOA for AH, and an epigenetic MOA for NTA. The MOA of PPX does not involve genotoxicity and may be specific to the 1321 diet.

Inhibition of the hepatocarcinogenicity of aflatoxin B1 in rats by low levels of the phenolic antioxidants butylated hydroxyanisole and butylated hydroxytoluene.

The phenolic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were studied for inhibition of aflatoxin B1 (AFB1) hepatocarcinogenesis in male Fischer 344 rats. The antioxidants were administered at 5, 25, or 125 ppm in AIN-76A diet for 42 weeks. Beginning with week 2, 5 micrograms/kg of AFB1 was given by intragastric instillation three times a week for 40 weeks either alone or concurrently with BHA or BHT feeding. The development of hepatocellular altered foci (HAF) induced by AFB1, as indicators of hepatocarcinogenesis, was monitored using immunohistochemical staining for the placental form of glutathione S-transferase. By 16 weeks the multiplicity of foci was 1.97/cm2 of liver area in rats given only AFB1, and this increased to 4.11/cm2 at 24 weeks and to 10.60/cm2 at 32 weeks. At the final sacrifice at 42 weeks, the multiplicity of foci was 12.90/cm2 compared to 0.75/cm2 in untreated controls. In rats given antioxidants in addition to AFB1, the high dose of BHA reduced the multiplicity to 7.72/cm2 and the high dose of BHT reduced the multiplicity to 9.35/cm2. Lower levels did not reduce foci induction. Thus, in male rats under the conditions of this experiment, the level of 125 ppm of either BHA or BHT inhibited the initiation of hepatocarcinogenesis by AFB1. The BHA effect was slightly greater than that of BHT.

Butylated hydroxytoluene lacks the activity of phenobarbital in enhancing diethylnitrosamine-induced mouse liver carcinogenesis.

The effect of the food additive butylated hydroxytoluene (BHT) as an enhancer of liver carcinogenesis in mice was investigated. Liver carcinogenesis was initiated by intraperitoneal injection of diethylnitrosamine (DEN) in male B6C3F1 mice at 100 or 200 mumol/kg body weight once a week for 10 weeks (total exposure 1000 or 2000 mumol/kg body weight). After an exposure-free recovery interval of 4 weeks, groups of mice were fed either basal diet or diets containing either 5000 ppm BHT or 500 ppm phenobarbital (PB), as a positive control, for 24 weeks. Exposure to the initiating doses of DEN alone induced no liver foci at 10 weeks or at 14 weeks after the recovery period, but at termination at 38 weeks, foci and adenomas were present in a dose-related incidence. In the groups given BHT after DEN/recovery, the incidence and the multiplicity of liver foci and adenomas were not different from those in mice given only DEN/recovery, whereas, in the groups given PB after DEN, liver lesions were increased by 1.7-3.0-fold. In conclusion, BHT had no promoting or syncarcinogenic effect on DEN-induced mouse liver carcinogenesis, whereas under the same conditions, PB acted as an enhancer.

N-ethyl-N-nitrosourea induced brain tumors in rats monitored by nuclear magnetic resonance imaging, plasma proton nuclear magnetic resonance spectroscopy and microscopy.

Characteristic slow growing brain gliomas were induced in rats by a single subcutaneous injection of N-ethyl-N-nitrosourea (ENU) within 24 h of birth. A parallel control group of rats was injected with saline. Seven treated rats developed gliomas within 2 years. Periodic nuclear magnetic resonance imaging (MRI) of the brain in 3-mm slices at 1.5 Tesla and monthly plasma sampling for proton magnetic resonance spectroscopy (MRS) at 360 MHz were started 6 months after the injection of ENU. In the MRS experiments, the Fossel index, average of the line widths of the methylene and methyl peaks at 360 MHz, was determined from half-line widths of methyl and methylene peaks at 0.8 ppm and 1.3 ppm. In five of the ENU injected animals that developed histologically verified brain tumors, these were also observed by MRI without contrast agents. There was no consistent correlation between the imaged tumors and the Fossel index obtained through MRS during the course of the study where repeated observations were performed on individual animals, nor was there any consistent statistical difference in the Fossel index between ENU-treated and control animals. The results of this study demonstrate that slowly developing carcinogen-induced brain tumors in rats can be successfully and reliably monitored noninvasively by MRI but not by MRS of plasma.

Nonlinearities in 2-acetylaminofluorene exposure responses for genotoxic and epigenetic effects leading to initiation of carcinogenesis in rat liver.

The dose responses for several effects of low-level limited exposures to 2-acetylaminofluorene (AAF) in the livers of male Fischer 344 rats were measured and a subsequent phenobarbital tumor promotion regimen was used to manifest initiation of carcinogenesis. Three doses over a 10-fold range yielding cumulative total exposures of 0.126, 0.42, and 1.26 mmol AAF/kg body weight were achieved by daily intragastric instillation for up to 12 weeks with interim terminations. This was followed by 24 weeks administration of 500 ppm phenobarbital (PB) in the diet to promote liver tumor development. At 12 weeks at the end of AAF administration, all exposures produced adducts in liver DNA, measured by 32P postlabeling, and the level of adducts increased with exposure, except that the high exposure did not produce a dose proportional increase. Measurement of arylsulfotransferase activity, a key enzyme in the metabolic activation of AAF, revealed that in livers from the high exposure animals, the enzyme was inhibited. To assess for toxicity, the centrilobular zone of glutamine synthetase-positive hepatocytes was quantified immunohistochemically at 12 weeks. The area of the zone was reduced in the high exposure group and there was a trend to reduction in relationship to exposure. The two lower exposures to AAF produced no increase in cell proliferation, whereas the high exposure resulted in a marked increase, about 8-fold over controls. Initiation was assessed by induction of hepatocellular altered foci (HAF) that expressed the placental form of glutathione S-transferase. AAF induced HAF in the high exposure group, 9-fold at 8 weeks and 170-fold at 12 weeks compared to controls. In rats maintained on PB for 24 weeks after exposure, the multiplicity of HAF increased in controls and comparably in the low and mid exposure groups, but remained at the about the same high level in the high exposure group. The high exposure produced a substantial incidence of benign neoplasms by 12 weeks, and with promotion by 36 weeks, all rats developed hepatocellular neoplasia. In the mid exposure group, only one adenoma occurred at 36 weeks in 17 rats, while in the low exposure group, no liver tumor occurred in 23 rats. Thus, these findings document nonlinearities for some of the effects of AAF, with supralinear effects at the high exposure for cell proliferation and induction of HAF, and a no-observed-effect level for induction of promotable liver neoplasms at the lowest cumulative exposure of 0.126 mmol/kg, in spite of the formation of DNA adducts. We conclude that the effects of this DNA-reactive hepatocarcinogen leading to initiation exhibit nonlinearities and possible thresholds.

Hyperpigmentation of the skin associated with minocycline therapy.

Skin biopsy specimens and discolored fingernails from minocycline-treated patients were examined by light and electron microscopy, histochemistry, and energy-dispersive x-ray analysis. Both hyperpigmented and adjacent normally pigmented skin samples contained pigment-laden macrophages in the dermis, although these cells were more numerous in the hyperpigmented skin samples. Elemental analysis showed that both pigment deposits and stratum corneum of hyperpigmented skin samples contained iron and calcium. Discolored areas of fingernails from a minocycline-treated patient also contained iron and calcium. Both skin and nail discoloration were possibly due to the presence of an iron chelate of minocycline and/or quinoid derivatives of minocycline. The presence of iron-containing pigment in normal as well as hyperpigmented skin may have predisposed to formation of minocycline-associated pigment in these patients.

Inhibition by acetaminophen of intestinal cancer in rats induced by an aromatic amine similar to food mutagens.

The widely used analgesic acetaminophen (APAP) was studied in rats for its ability to inhibit intestinal carcinogenesis induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB), which was selected as the carcinogen because of its similarity to the heterocyclic amines formed during cooking and which are postulated to be involved in colon cancer in humans. APAP was fed to male F344 rats at 250 ppm, which is about 1/4 the human therapeutic dose and at 5000 ppm, which is about fivefold the human dose. DMAB was injected subcutaneously at 50 mg/kg body weight weekly for 20 weeks, to assure identical exposures to all animals, followed by 22 weeks of maintenance. The DMAB was an effective inducer of tumours in the small and large intestines, producing an average of 1.3 tumours per animal. Feeding of APAP began 2 weeks before DMAB administration and continued for 44 weeks. A 9% reduction in the number of colon tumours per rat cancer at the low dose and an 86% reduction at the high dose were found. Small intestinal tumour incidence was reduced at both doses. The number of multiple intestinal tumours per rat was reduced by 27% and 49% for the low and high doses, respectively. The dimensions of these neoplasms, especially those in the colon, were also reduced in both dose groups. Thus, APAP, even at a sub-therapeutic dose, inhibited intestinal carcinogenesis induced by DMAB. This allows us to speculate that the effects of low exposures to dietary carcinogens of the heterocyclic amine type could be inhibited by therapeutic doses of APAP.

Anticarcinogenicity of monocyclic phenolic compounds.

The synthetic monocyclic phenolics (MPs), acetaminophen (APAP), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) are antimutagenic or anticarcinogenic against a diversity of chemical carcinogens affecting a variety of tissues in experimental animals. In studies in this laboratory of the anticarcinogenicity of MPs, the focus has been on delineating efficacy at low levels of MPs that do not elicit adaptive or toxic responses. To accomplish this, we are studying anticarcinogenicity against the neoplastic initiating activity of lower doses of carcinogens than have previously been studied and which are closer to human environmental exposures. In these studies, we have investigated anticarcinogenicity of BHT against liver cancer in rats induced by either 2-acetylaminofluorene (AAF) or aflatoxin B1 (AFB1) and anticarcinogenicity of APAP against colon cancer induced in rats by 3,2'-dimethyl-4-aminobiphenyl (DMAB). BHA and BHT at 100-125 ppm in the diet inhibited the initiation phase of AAF and AFB1 hepatocarcinogenesis and therefore may act intracellularly to block effects of the carcinogen. Likewise, with APAP in colon anticarcinogenicity, at 1000 ppm it reduced DNA binding and exerted a cytoprotective effect against DMAB. Thus, APAP also shows evidence of producing a blocking effect. We conclude that these MPs appear to be anticarcinogenic through a mechanism different from that of most other chemopreventive agents, possibly involving interception of the reactive chemical species of the carcinogen. Accordingly, they have promise as cancer prophylactics, including in combination with agents operating through other mechanisms.

Protective effect of acetaminophen against colon cancer initiation effects of 3,2'-dimethyl-4-aminobiphenyl in rats.

A previous investigation demonstrated the anticarcinogenicity of acetaminophen (APAP) against colon carcinogenesis in rats induced by 3,2'-dimethyl-4-aminobiphenyl (DMAB). DMAB was selected as a structurally related surrogate for heterocyclic amines, formed during cooking of protein, which are believed to be involved in human colon cancer. The objective of the present study was to ascertain whether the early initiating effects of this colon carcinogen are inhibited by APAP. Six groups of male F344 rats were treated over a 6-week period as follows: (1) vehicle (corn oil) for 6 weeks; (2) APAP in the diet at 1000 ppm daily for 6 weeks; (3) 50 mg/kg DMAB by gavage once a week for the last 4 weeks; (4) 5 mg/kg DMAB as for (3); (5) 1000 ppm APAP for 6 weeks and 50 mg/kg DMAB for the last 4 weeks; and (6) 1000 ppm APAP and 5 mg/kg DMAB as for (5). Colonic tissue was within normal limits in the control and APAP groups. In the APAP only group, apical enterocytic hypertrophy and hyperaemia over the entire surface epithelium was present. In the high-dose DMAB group, in the lower third of the crypts, foci of enlarged glands with hypertrophic cells exhibiting karyomegaly and anisokaryosis (FHE) of 3+ degree of severity were evident in 100% of the animals. Also, there were increases in periglandular fibrocytes, matrix and mononuclear cells (PF). In the low-dose DMAB group both FHE and PF changes with the same degree of severity were reduced. In rats given the low dose of DMAB plus APAP, FHE and PF with the same degree of severity (3+) was absent. Both DMAB exposures increased significantly the replicating fraction of colonic enterocytes in an exposure-related fashion and the replicating fractions were significantly reduced by APAP. In 32P-postlabelling of colon, liver and urinary bladder DNA, high-dose DMAB produced 2-6 distinct dose-related spots reflecting DNA adducts. These spots were reduced or were no longer detectable in all three tissues when APAP was given 2 weeks before and during DMAB exposure. Using immunohistochemical detection of DMAB adducts in the colon, a dose-related colour intensity was present for both doses of DMAB. APAP reduced this by 94-fold. Thus, APAP produced a marked protective effect in colonic enterocytes against several parameters of neoplastic development by the carcinogen.

Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated rats.

The relationship between thymic atrophy and plasma corticosterone levels was examined in 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-treated, pair-fed and ad libitum-fed male Sprague-Dawley rats given a usually lethal (125 micrograms/kg) or non-lethal (25 micrograms/kg) dose of TCDD. At both dosages, corticosterone levels in TCDD-treated animals begun to rise as early as day 4 after treatment. At later time points corticosterone levels were 5-7 times higher in rats given the non-lethal dose, and 6-10 times higher in rats administered the lethal dose than the levels observed in ad libitum-fed controls. Corticosterone levels in control rats pair-fed to the lethal dose group (as a result of the severe reduction in feed intake) were similarly elevated as in TCDD-treated rats but this was not the case in pair-fed rats of the non-lethal TCDD dosage (due to an essentially unchanged feed intake). At both dosages, relative thymus weights of TCDD-treated rats started decreasing by day 4 and continued to decline for the most part of the study. Relative thymus weights of rats pair-fed to the non-lethal TCDD dosage were not different from ad libitum-fed rats. However, the decrease in relative thymus weights of rats pair-fed to the lethal TCDD dosage paralleled that of TCDD-treated rats with an apparent 8-day lag period. Morphologically, the thymus as well as the adrenal revealed differential changes in TCDD-treated rats from those observable in pair-fed rats. These results suggest that either TCDD exerts a direct effect on the thymus and the adrenals or it causes an additional stress (e.g., a metabolic stress) over and above the starvation stress, which may be responsible for the differential morphological changes in these glands.

Chemically Induced Hepatocellular Proliferative Changes in the Rat Without Evidence of Neoplastic Transformation.

The hypothesis that hepatocellular proliferative changes give rise to autonomous, neoplastic lesions in rodents was tested in this study in which hepatoproliferative lesions induced in rats by feeding an experimental psychoactive drug, cyproximide, were examined at various times during the course of a 24 month study. A total of 610 male and female rats (Charles River Laboratories COBS®CD®(SD)BR, Wilmington, Mass.) were distributed into three groups. Each of two treatment groups contained 230 rats given 0.1% or 0.4% of cyproximide in the diet. One hundred and fifty rats were given a drug-free diet and served as controls. Five males and five females from each group were sacrificed for postmortem examination after 6, 12 and 20 months of drug diet feeding after which remaining rats were retained for recovery (nontreatment) periods of 18, 12 and 4 months, respectively. An additional 25 males and 25 females from each dose level were treated for 24 months and then sacrificed along with all surviving recovery and control rats. The results of this study demonstrated that the incidence of proliferative lesions was greater in the liver of treated rats (especially females) than in control rats; however, the incidence of hepatocellular neoplasms was the same in treated and control rats.

Safety assessment of butylated hydroxyanisole and butylated hydroxytoluene as antioxidant food additives.

Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are widely used antioxidant food additives. They have been extensively studied for potential toxicities. This review details experimental studies of genotoxicity and carcinogenicity which bear on cancer hazard assessment of exposure to humans. We conclude that BHA and BHT pose no cancer hazard and, to the contrary, may be anticarcinogenic at current levels of food additive use.

Toxicity studies of butylated hydroxyanisole and butylated hydroxytoluene. II. Chronic feeding studies.

The antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were fed in the diet to male F344 rats in two chronic feeding studies. In one study, feeding BHT for 76 wk at concentrations ranging from 100 to 6000 ppm produced no increase in neoplasms at any site. In a second study, feeding 12,000 ppm BHT for 110 wk had no neoplastic effect at any site, whereas feeding BHA at 12,000 ppm resulted in a small increase in squamous cell papillomas of the non-glandular squamous portion of the stomach.

Dose response of promotion by butylated hydroxyanisole in chemically initiated tumours of the rat forestomach.

The antioxidant food preservative butylated hydroxyanisole (BHA) was tested in an initiation-promotion protocol in which male F344 rats (6 wk old), 27 per group, were gavaged with a single dose of 200 mg N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)/kg. After 3 wk on control diet, test diets containing 0, 60, 300, 1000, 3000, 6000 or 12,000 ppm BHA were fed until termination of the experiment at approximately 110 wk, at which time most animals had died with stomach tumours. MNNG caused a high incidence of tumours in the glandular stomach and forestomach of all groups. Administration of 12,000 and 6000 ppm BHA, but not 3000 ppm or lower doses, caused statistically significant increases in the time-related incidence of MNNG-induced forestomach tumours as analyzed by life table analysis. BHA had no effect on the incidence of tumours in the glandular stomach or oesophagus. Tumour incidences in other organs were not related to BHA dose. No increase in hyperplasia in the oesophagus was evident in the high-dose BHA-treated animals compared with the MNNG-only group. This study provides corroboration that BHA affects only forestomach tumorigenesis and that the dose for enhancement of tumorigenesis is at least 1500-fold greater than human exposure.

Endocrine considerations in toxicologic pathology.

Detection of xenobiotic-induced toxicity on the endocrine system is a very difficult task because of the close relationship that the endocrine system has with the neural and immune systems. This is further complicated when one is asked to extrapolate from lab animals to man. Knowledge across species of hormonal action, solubility, transportation, plasma half life, receptor location, type of mediator, rhythmicity and pattern of secretion, is essential. One hormone can exert various effects in different tissues, or one function can be regulated by several hormones or even many functions of one endocrine target tissue can be regulated by several hormones acting in concert. The endocrine toxic response is determined by the state of differentiation of the target site. Feedback mechanisms both positive and negative, should also be taken into consideration initially. Because the effects of hormones have wide-ranged ramifications, the toxic responses likewise encompass broad areas such as the regulation of energy availability, maintenance of the internal environmental, development, growth and reproduction. The initial step involves the ascertaining of interference with the general trophic and target gland function and the characterization of the primary toxic effect. Equally important is to calculate the dose which elicited this primary effect, taking into consideration the area under the curve of the target endocrine site. Adapting this step-by-step approach, the causality between a specific toxic dose and a specific toxic effect can be readily and reliably established across all lab animal species and man.