The in vivo effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate on N-methyl-N-nitrosourea-induced apoptosis in mouse hair follicles.

This study assessed the in vivo relationship between apoptosis induced by the tumor initiator N-methyl-N-nitrosourea (MNU) and action of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in mouse hair follicle matrix cells. Mouse hair follicles were stimulated to grow hair synchronously by plucking resting hairs and MNU was applied to the plucked skin as the apoptosis inducer. The effects of TPA on MNU-induced apoptosis, when given at different intervals before or after MNU treatment, were examined. Changes in the percentage of apoptotic cells among total hair matrix cells after TPA treatment were measured. A significant suppression in levels of MNU-induced apoptosis was observed in the animals receiving TPA 1 to 6 hr following the induction. Administration of TPA before MNU caused a reduction in numbers of apoptotic cells over the control groups, but the differences were not significant. Determination of the diurnal variation in apoptotic levels in vehicle-treated mouse hair follicles revealed a relatively constant baseline pattern, suggesting that the above apoptotic responses to MNU and TPA were not affected by the background levels of apoptosis. The findings provided in vivo evidence which would support the hypothesis that TPA promotes tumorigenesis by preventing carcinogen-initiated cells from undergoing apoptotic death.

Role of lime in the generation of reactive oxygen species from betel-quid ingredients.

The role of lime in the formation of reactive oxygen species (ROS), i.e., O2-., H2O2, and OH., from betel-quid components (extracts of areca nut and catechu) was investigated in vitro using a chemiluminescence technique and an assay for oxidative DNA damage involving analysis of 8-hydroxy-2'-deoxyguanosine. Of the various areca-nut extracts, the catechin fraction, at alkaline pH, was shown to be the most active producer of ROS. The free Ca(OH)2 content and pH of lime samples (a component of betel quid and chewing tobacco) were highly correlated with the generation of ROS from areca-nut extract in vitro and with oxidative base damage to DNA in vitro. While Fe2+ had an enhancing effect on ROS formation, Mg2+ had a marked inhibitory effect. The cytogenetic effects of ROS generated in vivo were measured in Syrian golden hamsters in which the cheek pouch had been painted with lime and an areca-nut extract or catechu, singly or in combination. The frequency of micronucleated cells was increased only in animals that had received both the areca-nut extract and lime. The frequency of micronucleated cells in exfoliated oral mucosal cells from Indian chewers of betel quid with tobacco containing lime or of tobacco with lime was significantly higher than in a control (no habit) group. These studies demonstrate that addition of lime to betel quid constituents generates ROS, which induce cytogenetic damage in hamster cheek pouch and may contribute to the cytogenetic damage observed in the oral cavity of betel-quid chewers. These results implicate ROS in clastogenesis and probably in the etiology of oral cancer.

Dietary fibre, fat and beef modulation of colonic nuclear aberrations and microcapsule-trapped gastrointestinal metabolites of benzo[a]pyrene-treated C57/B6 mice consuming human diets.

Male C57/B6 mice were adapted to human diets of British origin that had 3-fold differences in either dietary fibre, fat or beef protein within the normal human range, and were then treated p.o. with 200 mg/kg benzo[a]pyrene (B[a]P) to induce colonic nuclear aberrations. [14C]B[a]P was included in the dose that followed 2 h after a gavage of magnetic PEI microcapsules. Untreated control groups were fed mouse chow or the baseline human diet which was low in all three dietary components (LLL). After the animals were killed at 24 h, large reductions (P less than 0.05) in colonic nuclear aberrations, and alterations to faecally excreted, microcapsule-trapped B[a]P metabolites were found for elevations of all three human diet components. Compared to untreated LLL control, B[a]P treatment gave an 8-fold increase in total nuclear aberrations, which was decreased 2- to 3-fold by increased fibre or fat. HPLC assay of B[a]P metabolites desorbed from microcapsules showed dietary fibre and beef protein to increase B[a]P diols and phenols but almost abolish B[a]P diones, consistent with a shift to enzymatic metabolism from non-specific oxidation. Increased fat considerably altered B[a]P metabolite disposition and microcapsule trapping, and comparison with microcapsules removed from colon contents indicated an altered enterohepatic circulation. Although it was not possible to attribute nuclear aberrations to individual B[a]P metabolites, a possible role of B[a]P diones seemed indicated, this being in line with previous microcapsule studies. These results show that microcapsules and human diets can be used in monitoring modulations of xenobiotic agents linked to mucosal chromosomal damage, with the eventual aim of human microcapsule biomonitoring.

A new type of lesion associated with severe fur damage in Canadian ranch foxes and an investigation of possible causes.

In the silver fox, as in its wild ancestor, the red fox (Vulpes vulpes L.), the annual growing phase (anagen) of guard hair follicles occupies at least four months. Severe damage to the hair coat near the end of this growing period was reported in 1985 on many ranches in New Brunswick and Nova Scotia. A histological analysis of serial sections of skin biopsies showed a marked increase in nuclear aberrations in the hair matrix of anagen guard hair follicles. These nuclear aberrations indicated that cells were undergoing apoptosis, a controlled form of cell death. Tissues from affected and unaffected foxes for histological and toxicological analysis, as well as other data, were obtained during visits to 26 ranches in 1986 and 34 ranches in 1987. Histological sections of the 1987 skin samples showed the mean percentage of nuclear aberrations in 43 unaffected foxes to be 0.08 +/- 0.01 (SEM), while that for 49 affected foxes was 0.51 +/- 0.23. The four foxes with the most severe coat damage also had the highest incidences of guard hair matrix cells with nuclear aberrations, ranging from 20 to 100 times greater than the mean for unaffected foxes. The mitotic index of the hair matrix, which normally remains fairly constant during the hair growth phase, was similar for unaffected and affected foxes (1.83 +/- 0.06 and 1.97 +/- 0.07 respectively). Although our analyses of field data have not established a specific environmental factor associated with increased nuclear aberrations, the possible involvement of toxic agents in follicle damage may warrant further investigation.

Assessment of 1,2-dimethylhydrazine in bone marrow micronucleus assay: variations in protocol and response.

The effect of variations in experimental protocol on the assessment of the genotoxicity of 1,2-dimethylhydrazine (DMH) in the bone marrow micronucleus assay was determined. The incidence of micronuclei (MN) in the bone marrow of CBA mice treated with DMH (either intraperitoneally (i.p.) or orally) was found to be significantly greater than that observed in C57B1/6J mice using the same dose and dosing regimen. With i.p. injection, DMH, at doses of 20 and 50 mg/kg, was found to be positive in the bone marrow MN test in CBA mice only. In C57B1/6J mice, DMH (i.p.) was found to be positive at only the 50 mg/kg dose. With oral administration, DMH was positive in the MN test only at the 50 mg/kg dose and only in CBA mice. No significant difference in the percentage of MN was observed when 300, 500, or 1,000 polychromatic erythrocytes (PCEs) were scored following a single treatment of DMH. Cyclophosphamide (CY) was found to induce a dose-dependent increase in the percentage of MN observed in the bone marrow of C57B1/6J mice. DMH tested positive in the colon nuclear aberration (NA) assay in both strains of mice using both i.p. and oral routes of administration, although C57B1/6J mice were found to be more sensitive than CBA mice. No significant difference was observed regarding the percentage of NAs observed in the colon between mice injected i.p. or orally gavaged.

Comparison of the activity of topically applied pesticides and the herbicide 2,4-D in two short-term in vivo assays of genotoxicity in the mouse.

Genotoxicity of eight topically applied compounds was determined using the bone marrow micronucleus (MN) test and hair follicle nuclear aberration (NA) assay in CD1 mice. Twenty-four hours after a single treatment, cyclophosphamide (CY), applied at doses corresponding to 1/4, 1/8, 1/16, and 1/32 of the published dermal LD50, and N-methyl-N-nitrosourea (MNU), applied at 1/4, 1/8, and 1/16 of the published dermal LD50, were found to increase the incidence of NA in a dose-dependent manner. The frequency of MN was significantly increased only at the highest dose of CY. Using the same protocol, six pesticides applied in dimethyl sulfoxide (DMSO) at doses of 1/8, 1/16, and 1/32 of the dermal LD50 were investigated. Aminocarb and chlordane induced a dose-dependent increase in the frequency of NA, while there was an observed increase in NA incidence at only the highest doses of dichlorvos (DDVP), 4,4'-DDT (DDT), and 2,4-dichlorophenoxyacetic acid (2,4-D). No effect was observed with fenitrothion on nuclear aberrations in hair follicles. Except for the highest dose of chlordane, none of the pesticides tested positive in the bone marrow micronucleus test. Serum cholinesterase levels were reduced to 70 +/- 4.7% of the DMSO control level with DDVP, 57 +/- 8.2% with aminocarb, and 60.3 +/- 4.8% with fenitrothion, indicating some systemic activity with these topically applied agents. The data suggest that aminocarb, chlordane, DDVP, DDT, and 2,4-D are genotoxic as determined by the NA assay and that this assay may be more useful in detecting topically applied genotoxic agents than the more often used bone marrow micronucleus test.

Nuclear aberrations in hair follicle cells of patients receiving cyclophosphamide. A possible in vivo assay for human exposure to genotoxic agents.

The toxic effect of cyclophosphamide on the proliferative cell population of hair follicles plucked from the human scalp was examined by the in vivo nuclear aberration assay. Patients participating in an independent clinical trial received oral low dose cyclophosphamide, intravenous high dose cyclophosphamide or oral placebo treatment. The percent of cells with nuclear aberrations (indicating apoptosis, a special form of cell death) and the percent of mitotic cells, in the hair matrix, were calculated for each patient before treatment and at several time points following cyclophosphamide or placebo treatment. The mean percentages of nuclear aberrations in both the treated Low dose and High dose cyclophosphamide patients were significantly higher than those for the pre-treatment and Placebo patients. The nuclear aberrations in hair follicle cells increased from pre-treatment (and Placebo) to treated Low dose and finally to treated High dose patients. The average percentage for pre-treatment samples from all patients was 0.06 +/- 0.03 SE. For 1 week and 1 month samples from Low dose patients it was 0.35 +/- 0.08 SE, and for combined 2,3 and 4 day samples from High dose patients it was 1.08 +/- 0.12 SE. Cyclophosphamide also had a significant effect on mitosis. A decrease in mitotic activity was observed at 1 month following the initial low dose cyclophosphamide treatment and at 24 +/- 2 h following each of the first two high dose cyclophosphamide treatments. The observed increase in nuclear aberrations following low dose as well as high dose cyclophosphamide suggests that it is feasible to use the nuclear aberration assay for in vivo human genotoxicity testing, using proliferating hair follicle cells.

Cardiopulmonary effects of a ketamine/acepromazine combination in hypovolemic cats.

The cardiopulmonary effects of a ketamine/ acepromazine combination was studied in ten cats subjected to a 25% whole blood volume loss. Test parameters included cardiac output, measured via thermodilution, heart rate, respiratory rate, arterial blood pressure (systolic, diastolic and mean) and blood gas analysis. Values for cardiac index, stroke volume and systemic vascular resistance were calculated from these data. Posthemorrhage, cardiac output, cardiac index, stroke volume, heart rate and measurements of arterial blood pressure were significantly decreased (p less than 0.05). Following the induction of ketamine/ acepromazine anesthesia, cardiac output, cardiac index, stroke volume and heart rate showed mild but statistically insignificant declines and were above their respective posthemorrhage values 120 min into ketamine/ acepromazine anesthesia. Measurements of arterial blood pressure showed further declines from their respective posthemorrhage values that were statistically significant (p less than 0.05). Following hemorrhage, respiratory rate increased significantly (p less than 0.05), associated with a fall in arterial CO2 tension. During ketamine/ acepromazine anesthesia, respiratory rate showed a dramatic and significant decline (p less than 0.05) with arterial CO2 tension rising to prehemorrhage values. Systemic vascular resistance, arterial O2 tension and pH remained essentially unchanged throughout the experimental period.

Cardiopulmonary effects of a halothane/oxygen combination in hypovolemic cats.

The cardiopulmonary effects of a halothane/oxygen combination were studied in eight cats subjected to a 25% whole blood volume loss. Test parameters included cardiac output measured via thermodilution, heart rate, respiratory rate, arterial blood pressure (systolic, diastolic and mean) and blood gas analysis. Values for cardiac index, stroke volume and systemic vascular resistance were calculated from these data. Posthemorrhage cardiac output, cardiac index, stroke volume and measurements of arterial blood pressure were significantly decreased (p less than 0.05). Heart rate remained unchanged. Following induction of halothane anesthesia the above parameters experienced a further significant decline (p less than 0.05) from their immediate preanesthetic (i.e. posthemorrhage) values. Heart rate also significantly decreased (p less than 0.05). Thirty minutes following the cessation of halothane anesthesia these values returned to near-hemorrhage levels, being above their respective preanesthetic values. Systemic vascular resistance initially rose, peaking ten minutes into halothane anesthesia, before gradually falling to prehemorrhage values at the end of halothane anesthesia. Following hemorrhage, respiratory rate demonstrated a transient increase, associated with an arterial CO2 tension fall, before returning to initial values at the preanesthetic time. During halothane anesthesia respiratory rate remained unchanged whereas arterial CO2 tension rose significantly (p less than 0.05) and pH declined slightly from preanesthetic readings. These returned to prehemorrhage values 30 minutes following the cessation of halothane anesthesia.

Nongenotoxicity of minoxidil in murine hair follicles as determined by the nuclear aberration assay.

A 1-cm2 area on the back of CD1 mice was prepared for topical application of minoxidil, N-methyl-N-nitrosourea (MNU), or cyclophosphamide (CY) by clipping or plucking hair from a patch of skin. Plucking stimulates hair follicle cell division while clipping does not. Minoxidil was topically administered for 8 consecutive days. CY or MNU was administered topically once on the eighth day postplucking. The incidence of nuclear aberrations and mitotic figures were measured in hair follicles while frequency of micronuclei and the ratio of RBC/PCE were measured in the bone marrow. Results with minoxidil showed no increase in either nuclear aberrations in the hair follicle or micronuclei in the bone marrow. These results suggest that topically applied minoxidil is not genotoxic. In contrast, a dose-dependent effect of MNU on the incidence of nuclear aberrations in the hair follicle was seen. CY induced a dose-dependent increase in the incidence of micronuclei in the bone marrow and in nuclear aberrations in the hair follicle after topical application. Minoxidil applied to clipped mice significantly increased the incidence of mitotic figures above that seen in both the clipped and plucked controls. This suggests that minoxidil is a mitogenic agent in the hair follicle. These findings are consistent with the success of topically applied minoxidil in the treatment of alopecia areata.

Inhibition of hepatocarcinogenic responses to 1,2-dimethylhydrazine by diallyl sulfide, a component of garlic oil.

The mechanisms by which diallyl sulfide (DAS), a component of garlic oil, inhibits hepatocarcinogenicity of 1,2-dimethylhydrazine (DMH) were examined in male Fischer 344 rats. Rats were subjected to partial hepatectomy to stimulate hepatocellular proliferation required for initiation by DMH (50-200 mg/kg i.p.) given 12 h later. Initiation was assessed by the numbers of foci and nodules of hepatocytes that were positive for gamma-glutamyl transpeptidase (gamma-GT) or glutathione-S-transferase-P (GST-P) after 6 weeks promotion by orotic acid (1% in semi-purified diet). DAS at doses above 50 mg/kg (by gavage) administered 1 h before DMH (50 mg/kg) partially reduced the numbers of gamma-GT and GST-P-positive foci. By comparison, all doses of DAS (25-100 mg/kg) completely prevented liver necrosis by DMH (200 mg/kg). DAS substantially reduced macromolecular binding of [14C]DMH in cultured liver cells, but had no effect on their levels of glutathione-S-transferase, glutathione reductase or glutathione peroxidase at 18 h. These findings suggest that low dosages of DAS which reduce DMH binding appear more likely to inhibit hepatocarcinogenicity by reducing the promoting influences of post-necrotic regeneration than by preventing initiation.

Studies on the mechanism of action of diallyl sulfide, an inhibitor of the genotoxic effects of cyclophosphamide.

Diallyl sulfide, a component of garlic oil, has been previously shown to inhibit induction of nuclear aberrations in the colons of mice treated with 1,2-dimethylhydrazine, a colon carcinogen. The ability of this agent to block cyclophosphamide-induced nuclear aberrations in urinary bladder and hair follicles was tested. [14C]Cyclophosphamide was injected and urine was collected to determine the disposition of metabolites of cyclophosphamide in mice treated with diallyl sulfide. This agent was capable of blocking nuclear aberration induction by cyclophosphamide in bladder and hair follicles. The effect was not mediated through inhibition of mitosis as determined by [3H]thymidine autoradiography in hair follicles. Diallyl sulfide pretreatment decreased the amount of radioactivity excreted in the urine in the first 24 h following cyclophosphamide treatment and blocked the appearance of acrolein, a cytotoxic metabolite of cyclophosphamide, in the urine over this time period. These results suggest that diallyl sulfide acts by conjugating the toxic metabolites of cyclophosphamide, thereby limiting their systemic circulation and diverting their route of excretion from the urine.

Lack of induction of nuclear aberrations by captan in mouse duodenum.

The fungicide, captan, is known to induce point mutations in vitro. In extremely high doses, technical grade captan leads to duodenal tumors in mice. In short-term in vivo assays for genotoxicity, equivocal results have been obtained with captan. In this study, the genotoxicity of captan was studied in vivo in the proximal small intestine of the mouse, the site of its oncogenicity. Orally administered captan up to 4,000 mg/kg body weight failed to induce a response in the small intestine nuclear aberration assay in a wide range of doses under a variety of experimental conditions, including pretreatment of animals with L-buthionine-S, R-sulfoximine (an inhibitor of glutathione biosynthesis). 1,2,3,6-Tetrahydrophthalimide and bis (trichloromethyl)disulfide, two compounds that have been identified as impurities in technical grade captan, also failed to produce positive results in this assay.

An in vivo assay for small intestine genotoxicity.

The induction of nuclear aberrations (NA) (apoptotic bodies and micronuclei) in duodenal crypts in a dose-dependent manner was associated with administration of agents known to induce tumours in the small intestine. These included X-irradiation, N-methyl-N-nitrosourea (MNU), benzo[a]pyrene (B[a]P), and 1,2-dimethylhydrazine (DMH), which were found to induce NA in cells in the proliferative region of crypts 24 h after they were given to mice. Methylurea (MU) and benzo[e]pyrene (B[e]P), which are non-carcinogenic structural analogues of MNU and B[a]P, respectively, did not induce NA under similar conditions. Based on these results, the ability of an agent to induce NA in the small intestine appears to reflect of its oncogenic potential in that organ.

Detection of nuclear anomalies in the colonic epithelium of the mouse.

Colon carcinogens produce a variety of nuclear anomalies including pyknotic, karyorrhectic, and micronucleated cells in the colonic epithelium within a few hr. Two model carcinogens, 1,2-dimethylhydrazine and gamma-rays, have been used to determine appropriate techniques, conditions, and scoring criteria for detecting such nuclear anomalies most efficiently. The results show that a rapid and sensitive assay for nuclear anomalies can be conducted with a variety of preparation techniques. We anticipate that the assay will be useful as a screen for potential colon carcinogens in the diet or elsewhere. The assay as recommended by us requires at least five animals per group and takes about 1 hr to analyze. A single sample at 6 or 24 hr after treatment should detect most carcinogens.

Pyrvinium pamoate lacks in vivo genotoxicity in the colon.

Pyrvinium pamoate, a widely used anthelmintic drug, has been previously tested for genotoxicity with equivocal results. Assays with bacterial or yeast test strains yielded positive results while in vitro tests with mammalian cells yielded negative results. In this study, the genotoxicity of pyrvinium was studied in vivo in the mouse colon, the therapeutic site of action of this agent. 1,2-Dimethylhydrazine (DMH), a colon carcinogen, was tested simultaneously as a positive control. The colonic nuclear aberration assay was used to determine genotoxicity. Pyrvinium delivered orally in three vehicles was not genotoxic in the murine colon, even at doses up to 12.5 times the recommended human dosage.

Nuclear aberrations as a short-term test for genotoxicity to the colon: evaluation of nineteen agents in mice.

The genotoxicity of 16 agents including several hydrazines, nitrosamines, aromatic amines, polycyclic hydrocarbons, and other related compounds and three known inhibitors of carcinogenesis was assessed in the murine colonic nuclear aberration assay. Of the seven agents considered positive for colonic DNA damage, five were large bowel carcinogens. All structural analogues of the intestinal carcinogens that are tumorigenic for other organs, with the exception of benzo[a]pyrene, were negative in the colonic nuclear aberration assay as were all noncarcinogens tested. The metabolic inhibitor disulfiram completely inhibited 1,2-dimethylhydrazine-induced colonic nuclear damage, while inhibition was less marked for the antioxidants butylated hydroxyanisole and caffeic acid. The versatility of the assay as an indicator of colonic genotoxicity resulting from carcinogen exposure is discussed.