Local carcinogenicity, rates of absorption, extent and persistence of macromolecular binding, and acute histopathological effects of N-hydroxy-1-naphthylamine and N-hydroxy-2-naphthylamine.

The N-hydroxy derivatives of 1- and 2-naphthylamine (NA) are directly carcinogenic at sites of application. In this study, the carcinogenicity of these two compounds at s.c. injection sites was compared with their relative rates of absorption, with the extent and persistence of their binding to protein, RNA, and DNA in the skin-subcutis, and with acute histopathological changes observed after local application. Male Sprague-Dawley rats were given injections of the N-hydroxy derivatives in the right rear leg at 16 mumol/dose. When administered twice weekly for 12 weeks, N-hydroxy-1-NA caused a 100% incidence (30 of 30) of poorly differentiated sarcomas at the injection site. N-Hydroxy-2-NA administered in a similar manner resulted in a low yield of tumors (7%; 2 of 30). Injection of N-hydroxy-1-NA once weekly for 12 weeks or twice weekly for 6 weeks also induced a high incidence of sarcomas (93 to 97%), but the time to tumor formation was significantly longer (p less than 0.0001) than in animals treated twice weekly for 12 weeks. The tumors were classified as malignant fibrous histiocytomas. Possible antagonistic or synergistic effects between the two compounds were also investigated. A sequential 6-week treatment with each of the N-hydroxy derivatives did not alter the expected tumor yields. However, alternating injections over 12 weeks caused a significant lengthening in the time to tumor formation (p less than 0.05). N-Hydroxy-1-NA bound covalently to protein, RNA, and DNA to a much greater extent than did N-hydroxy-2-NA. Protein binding with both derivatives decreased by 80 to 90% by 7 days after treatment. RNA binding in N-hydroxy-1-NA-treated rats markedly decreased, while N-hydroxy-2-NA-bound residues diminished only slightly. During this period, the extent of DNA binding with both derivatives remained fairly constant. When N-hydroxy-2-NA was injected 3 days after N-hydroxy-1-NA, there was a marked reduction in the apparent levels of N-hydroxy-1-NA bound to RNA and DNA. The greater tumorigenicity of N-hydroxy-1-NA versus N-hydroxy-2-NA correlated with its greater extent of macromolecular binding. Examination of acute histopathological changes occurring after single injections of N-hydroxy-1-NA and/or N-hydroxy-2-NA indicated that both compounds caused extensive necrosis in tissues at the injection site.(ABSTRACT TRUNCATED AT 400 WORDS)

Intestinal response to 1 alpha, 25-dihydroxycholecalciferol. I. RNA polymerase, alkaline phosphatase, calcium and phosphorus uptake in vitro, and in vivo calcium transport and accumulation.

The dynamics of intestinal response in rachitic chicks to 1alpha,25-dihydroxycholecalciferol were evaluated by various biochemical parameters. The following observations were made: 1. The earliest detected intestinal response to 1alpha,25-dihydroxycholecalciferol was increased in vitro calcium uptake and in vivo calcium transport, occurring by 2 h and 2.5 h respectively. 2. Increased RNA polymerase activity was observed by 4 h after 1alpha,25-dihydroxycholecalciferol treatment. 3. Calcium binding protein was detected by 5 h, but could not be detected 2.5 h after 1alpha,25-dihydroxycholecalciferol treatment. 4. Increased alkaline phosphatase activity and in vitro accumulation of inorganic phosphate were first demonstrable 6 h after 1alpha,25-dihydroxycholecalciferol treatment. 5. In vivo duodenal calcium accumulation in the mucosa was elevated after 5 h, peaked at 6.5 h, and then began to decrease at 9 h. In vitro duodenal calcium accumulation was elevated at 2 h, peaked at 12 h, and decreased to control level by 18 h. Our data emphasize the lack of correlation between the appearance of calcium binding protein or increased alkaline phosphatase activity and the transport rate of calcium across the duodenum after treatment with 1alpha,25-dihydroxycholecalciferol. The data suggest a correlation between duodenal calcium accumulation and the appearance of calcium binding protein or increased alkaline phosphatase activity.

Immunoperoxidase localization of vitamin D dependent calcium binding protein.

Calcium binding protein (CaBP) was localized by the indirect peroxidase-labeled antibody method in chick duodenum 72 hr after administering 32.5 nmol of cholecalciferol to vitamin D-deficient chicks. CaBP was observed in cytoplasm and nuclei of absorptive cells but was absent from goblet cells. Our results are consistent with the suggested functional role for CaBP in the prevention of intracellular accumulation of calcium by preventing mitochondrial accumulation of calcium, enhancing removal of calcium from absorptive cells, and/or preventing the "leaking" of calcium into cells through the lateral borders. They are not consistent with an extracellular functional role for CaBP.

Susceptibility of C57BL/6 mice to tumorigenicity induced by dimethylnitrosamine and 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine in the neonatal bioassay.

Male C57BL/6 neonates were treated on days 8 and 15 with 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 6.5 or 26.2 mg/kg) or dimethylnitrosamine (DMN, 2.6 or 10.5 mg/kg). No tumors were seen in PhIP-treated animals at 15 months of age. Liver and lung tumor incidences in DMN-treated animals were 67-79 and 0-7%, respectively. In comparison with data from other strains, our results indicate that (1) neonatally-treated C57BL/6 mice are resistant to the induction of liver and lung tumors by PhIP and lung tumors by DMN and (2) the susceptibility of this strain to induced liver tumors correlates with the activity of hepatic DMN N-demethylase and PhIP N-hydroxylase in the (untreated) neonates.

Inhibitory effect of caloric restriction on tumorigenicity induced by 4-aminobiphenyl and 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) in the CD1 newborn mouse bioassay.

The tumorigenicity of 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) were studied in combination with caloric restriction in the male neonatal CD1 mouse bioassay. 4-ABP and PhIP exhibited moderate and weak tumorigenicity, respectively, in ad libitum fed mice; however, none of the caloric restricted mice developed tumors. These results indicate that caloric restriction, even when begun 3 months after the conclusion of compound treatment, markedly inhibited 4-ABP- and PhIP-induced tumors in the CD1 mouse.

Evaluation of the transgenic p53+/- mouse for detecting genotoxic liver carcinogens in a short-term bioassay.

The transgenic p53-deficient heterozygous (p53+/-) mouse is prone to both spontaneous and induced tumors and has been proposed for use in a sensitive, short-term (6 months) assay for identifying genotoxic, multispecies carcinogens. It is not clear, however, if a short-term assay with p53+/- mice detects agents that target certain organs, in particular, the liver. In this study, we treated neonatal male p53+/- and p53+/+ mice with the genotoxic carcinogens dimethylnitrosamine (DMN), 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP), and 6-nitrochrysene (6-NC). In keeping with the methodology of the proposed short-term assay, the p53+/- mice were evaluated for tumors 7 months after treatment. Wild-type neonatal mice treated with genotoxic carcinogens are known to develop tumors within 1 year; hence, the p53+/+ animals used as controls were subjected to pathological examination at 1 year of age. Our results showed that PhIP was not tumorigenic in either group of mice. Liver tumor incidence increased significantly in the p53+/+ mice treated with DMN and 6-NC, indicating that the conditions of the bioassay were conducive to the promotion of liver tumorigenesis. On the other hand, these two chemicals failed to induce a significant increase in liver tumors in the p53+/- mice by seven months. This result suggests that a deficiency in the amount of p53 protein does not lead to accelerated liver tumorigenesis in mice, and contrasts with previous reports that show a decreased latency of tumors in non-liver targets.

Tumorigenicity of nitropolycyclic aromatic hydrocarbons in the neonatal B6C3F1 mouse bioassay and characterization of ras mutations in liver tumors from treated mice.

The nitropolycyclic aromatic hydrocarbons (nitro-PAHs) 1-, 2-, and 3-nitrobenzo[a]pyrene, 1- and 3-nitrobenzo[e]pyrene, 2- and 3-nitrofluoranthene, 9-nitrodibenz[a,c]anthracene, and two of the parent PAHs fluoranthene and dibenz[a,c]anthracene were tested for tumorigenicity in the neonatal male B6C3F1 mouse. 6-Nitrochrysene was used as a positive control. Mice were administered three intraperitoneal injections of test agent (400 nmol total) on 1, 8, and 15 days after birth and evaluated for liver and lung tumors at 12 months of age. 2-Nitrobenzo[a]pyrene and 6-nitrochrysene induced a high incidence of liver tumors (91-100%), while the remaining test compounds did not induce tumors at a rate significantly higher than the solvent control. 6-Nitrochrysene was the only test agent to produce a significant increase in the frequency of lung tumors. K- and H-ras mutations were analyzed in liver tumors of treated mice and mainly occurred at the first base of K-ras codon 13, resulting in GGC --> CGC transversion. Since most of the tested nitro-PAHs are mutagens in vitro, the results of this study indicate that the in vitro mutagenicity of these compounds does not correlate with their tumorigenicity in the neonatal B6C3F1 mouse bioassay. Also, the results indicate that liver tumors from mice treated with nitro-PAHs possess ras mutations typical of PAHs and their derivatives.

Fumonisin b1 carcinogenicity in a two-year feeding study using F344 rats and B6C3F1 mice.

Fumonisin B1 (FB1) is a mycotoxin isolated from Fusarium fungi that contaminate crops worldwide. A previous study demonstrated that FB1 promoted preneoplastic foci in initiated rats and induced hepatocellular carcinomas in BD IX rats at 50 parts per million (ppm), but fundamental dose-response data were not available to assist in setting regulatory guidelines for this mycotoxin. To provide this information, female and male F344/N/Nctr BR rats and B6C3F1 Nctr BR mice were fed for two years a powdered NIH-31 diet containing the following concentrations of FB1: female rats, 0, 5, 15, 50, and 100 ppm; male rats, 0, 5, 15, 50, and 150 ppm; female mice, 0, 5, 15, 50, and 80 ppm; male mice, 0, 5, 15, 80, and 150 ppm. FB1 was not tumorigenic in female F344 rats with doses as high as 100 ppm. Including FB1 in the diets of male rats induced renal tubule adenomas and carcinomas in 0/48, 0/40, 9/48, and 15/48 rats at 0, 5, 15, 50, and 150 ppm, respectively. Including up to 150 ppm FB1 in the diet of male mice did not affect tumor incidence. Hepatocellular adenomas and carcinomas were induced by FB1 in the female mice, occurring in 5/47, 3/48, 1/48, 19/47, and 39/45 female mice that consumed diets containing 0, 5, 15, 50, and 80 ppm FB1, respectively. This study demonstrates that FB1 is a rodent carcinogen that induces renal tubule tumors in male F344 rats and hepatic tumors in female B6C3F1 mice.

Compensatory regeneration as a mechanism for renal tubule carcinogenesis of fumonisin B1 in the F344/N/Nctr BR rat.

Fumonisin B1(FB1) is a fungal metabolite of Fusarium verticillioides (= F. moniliforme), a fungus that grows on many crops worldwide. Previous studies demonstrated that male BD IX rats consuming diets containing 50 ppm fumonisin B1 developed hepatocellular carcinomas. In our recent studies, diets containing FB1 at 50 ppm or higher concentrations induced renal tubule carcinomas in male F344/N/Nctr BR rats and hepatocellular carcinomas in female B6C3F1/Nctr BR mice. The carcinogenicity of FB1 in rats and mice is not due to DNA damage, as several laboratories have demonstrated that FB1 is not a genotoxin. FB1 induces apoptosis in cells in vitro. Including FB1 in the diets of rats results in increased hepatocellular and renal tubule epithelial cell apoptosis. In studies with F344/N/Nctr BR rats consuming diets containing up to 484 ppm FB1 for 28 days, female rats demonstrated more sensitivity than male rats in the induction of hepatocellular apoptosis and mitosis. Conversely, induction of renal tubule apoptosis and regeneration were more pronounced in male than in female rats. Induction of renal tubule apoptosis and hyperplasia correlated with the incidence of renal tubule carcinomas that developed in the 2-year feeding study with FB1 in the F344/N/Nctr BR rats. The data are consistent with the hypothesis that the induction of renal tubule carcinomas in male rats could be partly due to the continuous compensatory regeneration of renal tubule epithelial cells in response to the induction of apoptosis by fumonisin B1.

Temporal changes in tissue repair permit survival of diet-restricted rats from an acute lethal dose of thioacetamide.

Although, diet restriction (DR) has been shown to substantially increase longevity while reducing or delaying the onset of age-related diseases, little is known about the mechanisms underlying the beneficial effects of DR on acute toxic outcomes. An earlier study (S. K. Ramaiah et al., 1998, Toxicol. Appl. Pharmacol. 150, 12-21) revealed that a 35% DR compared to ad libitum (AL) feeding leads to a substantial increase in liver injury of thioacetamide (TA) at a low dose (50 mg/kg, i.p.). Higher liver injury was accompanied by enhanced survival. A prompt and enhanced tissue repair response in DR rats at the low dose (sixfold higher liver injury) occurred, whereas at equitoxic doses (50 mg/kg in DR and 600 mg/kg in AL rats) tissue repair in AL rats was substantially diminished and delayed. The extent of liver injury did not appear to be closely related to the extent of stimulated tissue repair response. The purpose of the present study was to investigate the time course (0-120 h) of liver injury and liver tissue repair at the high dose (600 mg TA/kg, i.p., lethal in AL rats) in AL and DR rats. Male Sprague-Dawley rats (225-275 g) were 35% diet restricted compared to their AL cohorts for 21 days and on day 22 they received a single dose of TA (600 mg/kg, i.p.). Liver injury was assessed by plasma ALT and by histopathological examination of liver sections. Tissue repair was assessed by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) immunohistochemistry during 0-120 h after TA injection. In AL-fed rats hepatic necrosis was evident at 12 h, peaked at 60 h, and persisted thereafter until mortality (3 to 6 days). Peak liver injury was approximately twofold higher in DR rats compared to that seen in AL rats. Hepatic necrosis was evident at 36 h, peaked at 48 h, persisted until 96 h, and returned to normal by 120 h. Light microscopy of liver sections revealed progression of hepatic injury in AL rats whereas injury regressed completely leading to recovery of DR rats by 120 h. Progression of injury led to 90% mortality in AL rats vs 30% mortality in DR group. In the surviving AL rats, S-phase DNA synthesis was evident at 60 h, peaked at 72 h, and declined to base level by 120 h, whereas in DR rats S-phase DNA synthesis was evident at 36 h and was consistently higher until 96 h reaching control levels by 120 h. PCNA studies showed a corresponding increase in cells in S and M phase in the AL and DR groups. DR resulted in abolition of the delay in tissue repair associated with the lethal dose of TA in ad libitum rats. Temporal changes and higher tissue repair response in DR rats (earlier and prolonged) are the conduits that allow a significant number of diet restricted rats to escape lethal consequence.

Antimitotic intervention with colchicine alters the outcome of o-DCB-induced hepatotoxicity in Fischer 344 rats.

Although, hepatotoxic injury of 1,2-dichlorobenzene (o-DCB) is greater in Fischer 344 (F344) as compared to Sprague-Dawley (S-D) rats, this interstrain difference does not transcend into any difference in lethal effects of o-DCB. Interstrain difference in compensatory tissue repair has been suggested as the underlying mechanism for the lack of strain differences in lethality (S.G. Kulkarni, H. Duong, R. Gomila, and H.M. Mehendale, Strain differences in tissue repair response to 1,2-dichlorobenzene. Archives of Toxicology 1996; 70: 714-723). If higher tissue repair in F344 rats compensates for more severe liver injury, then antimitotic intervention after infliction of o-DCB-induced liver injury should lead to lethality in F344 rats. Colchicine (CLC, 1 mg/kg) functions as an effective antimitotic agent and does not cause any side effects apart from suppressing cellular proliferation. Two groups of male F344 rats (160-190 g) received a single dose of 0.6 ml o-DCB/kg: 30 h later one group of rats received CLC (1 mg/kg; i.p.) and the other received distilled water (1 ml/kg; i.p.). Liver injury was assessed by measuring plasma ALT and SDH activity, liver histopathology, and liver regeneration was estimated by [3H]thymidine incorporation into hepatonuclear DNA and proliferating cell nuclear antigen (PCNA) assay in both groups. Similar liver injury was noted in both the o-DCB + vehicle and o-DCB + CLC treated F344 rats at 36 h indicating that CLC does not interfere with the uptake, bioactivation and causation of injury by o-DCB. S-phase synthesis which occurred at 36 h in the o-DCB + vehicle group was blocked in the o-DCB + CLC group. CLC administration 6 h prior to S-phase stimulation selectively abolished S-phase stimulation at 36 h, and led to 50% lethality. Since the effect of CLC antimitosis was transient, S-phase synthesis occurring at 48 h was not blocked and was sustained up to 72 h thereby allowing the other 50% of rats to overcome liver injury induced by o-DCB and survive the lethal outcome. These findings suggest that a significantly higher rate of compensatory tissue repair in F344 rats enables them to overcome more severe liver injury inflicted by o-DCB.

Stimulated pulmonary cell hyperplasia underlies resistance to alpha-naphthylthiourea.

The rodenticide alpha-naphthylthiourea (ANTU) causes pulmonary edema and pleural effusion that leads to death via pulmonary insufficiency. Rats become resistant to the lethal effect of ANTU if they are first exposed to a small, nonlethal dose of ANTU. Young rats are also resistant to ANTU. The mechanism by which rats develop resistance by a prior, small dose exposure has yet to be determined. Growth factor induced-pulmonary hyperplasia has been demonstrated to attenuate ANTU-induced lung leak. We hypothesized that a small dose of ANTU protects against a large dose through pulmonary cell hyperplasia induced by the protective dose. Furthermore, we hypothesized that this hyperplasia is associated with altered transcription of growth factors. Male Sprague-Dawley rats (175-225 g) were treated with a low dose of ANTU (5 mg ANTU/kg; ANTU(L)) 24 h before challenge with a 100% lethal dose of ANTU (70 mg ANTU/kg; ANTU(H)) resulting in 100% protection against the lethal effect of ANTU(H). ANTU(L) protection against ANTU(H) lasted for 5 days, slowly phased out, all being lost by day 20. Injury was assessed by estimating pulmonary vascular permeability and through histopathological examination. ANTU(H) alone resulted in an increase in pulmonary edema leading to animal death. However, injury was prevented if the rats were first treated with ANTU(L). There was a stimulation of pulmonary cell hyperplasia in the lungs of ANTU(L) treated rats as measured by [3H]-thymidine and bromodeoxyuridine incorporation. Treatment with the antimitotic agent colchicine abolished ANTU(L)-induced resistance to ANTU(H). ANTU resistant rats were also resistant to the lethal effect of paraquat. Paraquat is not taken up by pneumocytes if they are undergoing hyperplasia. ANTU(L) administration resulted in an up regulation of gene transcription for keratinocyte growth factor, transforming growth factor-beta, keratinocyte growth factor receptor and epidermal growth factor receptor as determined through reverse transcription-polymerase chain reaction. A significant increase in transforming growth factor-alpha was not observed. These findings collectively suggest that ANTU(L)-induced pulmonary cell hyperplasia underlies resistance to ANTU(H). Furthermore, the stimulation of hyperplasia may be due to altered growth factor and growth factor receptor expressions.

Deferoxamine delays the development of the hepatotoxicity of acetaminophen in mice.

The hepatotoxicity of acetaminophen is conventionally ascribed to metabolism by CYP450 to N-acetyl-p-benzoquinone imine and covalent binding to proteins. We investigated a potential role for oxidative stress by determining the effect of the ferric chelator deferoxamine (Desferal) on acetaminophen (paracetamol)-induced hepatotoxicity in mice. Administration of deferoxamine (75 mg/kg) 1 h after a toxic dose of acetaminophen (300 mg/kg) significantly delayed the development of the toxicity without altering covalent binding. In saline-treated mice serum ALT was 18 +/- 2 IU/l. In acetaminophen-treated mice serum alanine aminotransferase (ALT) was 779 +/- 271 at 2 h, 7421 +/- 552 IU/l at 4 h, 5732 +/- 523 IU/l at 8 h, and 5984 +/- 497 IU/l at 24 h. In acetaminophen plus deferoxamine-treated mice, serum ALT was 80 +/- 10 at 2 h, 472 +/- 74 IU/l at 4 h, 2149 +/- 597 IU/l at 8 h, and 5766 +/- 388 at 24 h. Deferoxamine at 1 h after acetaminophen did not decrease serum ALT at 12 h; however, deferoxamine at 1 and 4 h, or deferoxamine at 1 h plus N-acetylcysteine at 4 h to replete hepatic glutathione, decreased the toxicity from 5625 +/- 310 IU/l to 3436 +/- 546 IU/l and 3003 +/- 282 IU/l, respectively. Deferoxamine plus N-acetylcysteine at 1.25 h after acetaminophen was more effective at decreasing the 24 h toxicity than N-acetylcysteine alone. In acetaminophen treated mice, higher doses of deferoxamine (150-300 mg/kg) at 1 h greatly increased the observed hepatotoxicity at 4 h in a dose responsive manner, but deferoxamine alone was nontoxic.

Influence of sampling on the reproducibility of ovarian follicle counts in mouse toxicity studies.

Different ovarian follicle counting procedures were investigated to reduce labor while retaining statistical power. Intact ovaries of untreated CD-1 mice (20/group) from National Toxicology Program Reproductive Assessment by Continuous Breeding (RACB) studies were serially sectioned at 6 microm. Mean numbers of small and growing follicles were used to assess sampling efficiency. In 10 mice per group, comparisons were made between 10% nonrandom samples from every 10th section starting at either the first or sixth section having follicles (approximately 40 sections per ovary). These 10% counts were compared with 5% (20 sections) and 20% (80 sections) nonrandom samples and with 1% (4 sections), 5%, or 10% random samples from the same 10 animals. For two studies, a 10% nonrandom sample was analyzed from 20 mice per group. Follicle counts for each group were comparable regardless of the sampling paradigm. Four to 10 animals provided 90% confidence that a 20% difference in mean counts would be detected. The 1% sample had a larger error term and, thus, slightly reduced statistical power. These data suggest that follicle counts from 1% or 5% random samples may provide a suitable screen for ovarian toxicity.

Effects of dietary genistein exposure during development on male and female CD (Sprague-Dawley) rats.

Genistein is a naturally occurring isoflavone that interacts with estrogen receptors and multiple other molecular targets. Human exposure to genistein is predominantly through consumption of soy products, including soy-based infant formula and dietary supplements. A dose range-finding study was conducted as a prelude to a multigeneration bioassay to assess potential toxicities associated with genistein consumption. Genistein was administered in a soy- and alfalfa-free diet at 0, 5, 25, 100, 250, 625, or 1250 ppm to pregnant dams starting on Gestation day 7 and continuing throughout pregnancy. Dietary exposure of the dams continued through lactation, and pups were maintained on the same dosed feed as their mother after weaning until sacrifice at Postnatal day 50. Body weight and feed consumption of the treated dams prior to parturition showed a decreasing trend with a significant reduction at the highest dose. Litter birth weight was depressed in the 1250 ppm dose group, and pups of both sexes in that dose group had significantly decreased body weights relative to controls at the time of sacrifice. The most pronounced organ weight effects in the pups were decreased ventral prostate weight in males at the 1250 ppm dose and a trend toward higher pituitary gland to body weight ratios in both sexes. Histopathologic examination of female pups revealed ductal/alveolar hyperplasia of the mammary glands at 250 to 1250 ppm. Ductal/alveolar hyperplasia and hypertrophy also occurred in males, with significant effects seen at 25 ppm and above. Abnormal cellular maturation in the vagina was observed at 625 and 1250 ppm, and abnormal ovarian antral follicles were observed at 1250 ppm. In males, aberrant or delayed spermatogenesis in the seminiferous tubules relative to controls was observed at 1250 ppm. There was a deficit of sperm in the epididymis at 625 and 1250 ppm relative to controls, although testicular spermatid head counts and epididymal spermatozoa counts did not show significant differences from controls at these doses. Both sexes showed an increase in the incidence and/or severity of renal tubal mineralization at doses of 250 ppm and above. Dietary genistein thus produced effects in multiple estrogen-sensitive tissues in males and females that are generally consistent with its estrogenic activity. These effects occurred within exposure ranges achievable in humans.

The role of programmed cell death in the toxicity of the mutagens, ethyl methanesulfonate and N-ethyl-N'-nitrosourea, in AHH-1 human lymphoblastoid cells.

In order to determine the pathway for cell death in alkylating agent-exposed human lymphoblastoid cells, AHH-1 cells were exposed to either ethyl methanesulfonate (EMS) or ethyl nitrosourea (ENU) and the effect on relative cell growth and plating efficiency quantified. Flow cytometric (FCM) assays were utilized to quantify cell viability and to determine if cell death occurred through necrosis or apoptosis. As expected, exposure to the simple ethylating agents resulted in concentration-dependent decreases in plating efficiencies at each time interval after exposure (Days 0, 2, 3 and 7). EMS exposure did not significantly affect the relative cell growth, in contrast to ENU exposure, which inhibited cell growth. The FCM viability assay, based on light scatter characteristics, revealed that exposure to either alkylating agent resulted in a significant reduction in the percentage of viable cells. The results of the FCM dye-exclusion assays revealed that while necrosis occurred in EMS- and ENU-exposed cells, the primary manner of cell death was apoptosis. AHH-1 cells were stained with propidium iodide and fluorescein diacetate, the population of cells sorted electronically and the cell type (necrotic, apoptotic or viable) confirmed morphologically. Our results clearly indicate that exposure to EMS or ENU results in the movement of AHH-1 cells into the pathway for apoptosis and cell death.

The mycotoxin fumonisin induces apoptosis in cultured human cells and in livers and kidneys of rats.

Fumonisin B1 is a mycotoxin produced by Fusarium moniliforme, a fungus that infects corn and other grains in the U.S. Fumonisin ingestion causes a variety of effects including equine leukoencephalomalacia and porcine pulmonary edema, and has been associated epidemiologically with human esophageal cancer. Fumonisin B1 produces growth inhibition and increased apoptosis in primary human keratinocyte cultures and in HET-1A cells. In order to set the doses for a 2-year tumor bioassay, male and female F344 rats were fed fumonisin B1 (99, 163, 234, and 484 ppm) for 28 days and the organs examined histologically. There was a dose dependent decrease in liver and kidney weights in the rats. The liver weight loss was accompanied by the induction of apoptosis and hepatocellular and bile duct hyperplasia in both sexes, with the female rats being more responsive at lower doses. The induction of tubular epithelial cell apoptosis was the primary response of the kidneys to dietary fumonisin B1. Apoptosis was present at all doses in the kidneys of the male rats, and occurred in the females only at 163, 234, and 484 ppm fumonisin B1. These results demonstrate that fumonisin B1 treatment causes a similar increase in apoptosis both in vivo and in vitro.

Hematologic and serum chemistry reference values for adult brown mink.

Hematologic and serum chemistry reference values were determined for 160 12-month-old brown untamed captive mink (Mustela vision). Blood was obtained by jugular venipuncture after administration of ketamine and xylazine. There were no statistically significant differences between male and female mink. The packed cell volume, hemoglobin, and red blood cell count were 10 to 20% lower than previously reported for non-anesthesized mink. Serum glucose, alanine aminotransferase and aspartate aminotransferase values also were lower than previously reported values.

Non-invasive antromycosis: (diagnosis and treatment).

Increasing numbers of cases of chronic maxillary sinusitis are encountered which resist frequent sinus irrigation and treatment of predisposing factors. The antral washouts are of unusual consistencies and colours. The antral washouts of six cases were investigated by culture and microscopic examination and proved to contain fungal colonies. Antroscopy was a valuable asset in diagnosis. One patient had a Caldwell-Luc operation and daily irrigation through an indwelling polythene tube followed by daily instillation of clotrimazole (Canesten). The five patients who refused operation were treated by repeated bi-weekly antral washouts followed by instillation of clotrimazole. Weekly samples of antral secretions were examined by culture and microscopic examination until they were free for four consecutive weeks. We believe that frequent antral irrigation and local instillation of a broad-spectrum antimycotic drug--preferably after debridement--is the treatment of choice for antromycosis.

Tonsillomycosis: a myco-histopathological study.

Tissues from 227 tonsillectomies, mainly in adults, were examined culturally and histopathologically for the presence of fungus. Actinomyces was demonstrated in 69 cases (30.40 per cent) by both methods. Yeasts and moulds were each isolated from 21 cases (9.25 per cent) and mixed isolates were obtained from 7 cases (3.08 per cent). There was no histopathological evidence of these organisms. Negative cultural and histopathological results were obtained in 53 cases (23.35 per cent). Chloramphenicol-resistant bacteria were cultured in 56 cases (25.11 per cent) but were seen histopathologically in only 25 cases (11.01 per cent). In all cases in which Actinomyces or bacteria were present histopathologically, a minimal tissue response indicated that these organisms were saprophytes and not primary pathogens.