Memory deterioration based on the tobacco smoke exposure and methylazoxymethanol acetate administration vs. aripiprazole, olanzapine and enrichment environment conditions.

Enrichment environment conditions, as well as tobacco smoke exposure, may affect cognitive function (e.g. spatial memory) in an animal model of schizophrenia and schizophrenic patients. The aim of this study was to find whether spatial memory function impairment is found in methylazoxymethanol acetate treated rats (an animal model of schizophrenia) and whether aripiprazole (1.5 mg/kg) and olanzapine (0.5 mg/kg) modify these functions. We also were able to determine whether tobacco smoke exposure and enrichment environment conditions have an impact on drug efficacy. The effect of methylazoxymethanol acetate, tobacco smoke exposure, enrichment environment and the use of drugs were studied in the Morris Water Maze test (spatial memory). The results of our study clearly show that enriched environment may have a procognitive effect while tobacco smoke and methylazoxymethanol acetate have a contradictory effect. This paper also confirmed that the use of neuroleptics, namely ARI and OLA, reduced the process of spatial memory deterioration tested in the Morris water maze both in terms of the number of escape latencies and crossed quadrants.

Time-matched analysis of DNA adduct formation and early gene expression as predictive tool for renal carcinogenesis in methylazoxymethanol acetate treated Eker rats.

Genotoxic carcinogens pose great hazard to human health. Uncertainty of current risk assessment strategies and long latency periods between first carcinogen exposure and diagnosis of tumors have raised interest in predictive biomarkers. Initial DNA adduct formation is a necessary step for genotoxin induced carcinogenesis. However, as DNA adducts not always translate into tumorigenesis, their predictive value is limited. Here we hypothesize that the combined analysis of pro-mutagenic DNA adducts along with time-matched gene expression changes could serve as a superior prediction tool for genotoxic carcinogenesis. Eker rats, heterozygous for the tuberous sclerosis (Tsc2) tumor suppressor gene and thus highly susceptible towards genotoxic renal carcinogens, were continuously treated with the DNA alkylating carcinogen methylazoxymethanol acetate (MAMAc). Two weeks of MAMAc treatment resulted in a time-dependent increase of O6-methylguanine and N7-methylguanine adducts in the kidney cortex, which was however not reflected by significant expression changes of cyto-protective genes involved in DNA repair, cell cycle arrest or apoptosis. Instead, we found a transcriptional regulation of genes involved in the tumor-related MAPK, FoxO and TGF-beta pathways. Continuous MAMAc treatment for up to 6 months resulted in a mild but significant increase of cancerous lesions. In summary, the combined analysis of DNA adducts and early gene expression changes could serve as a suitable predictive tool for genotoxicant-induced carcinogenesis.

Enrichment and training improve cognition in rats with cortical malformations.

Children with malformations of cortical development (MCD) frequently have associated cognitive impairments which reduce quality of life. We hypothesized that cognitive deficits associated with MCD can be improved with environmental manipulation or additional training. The E17 methylazoxymethanol acetate (MAM) exposure model bears many anatomical hallmarks seen in human MCDs as well as similar behavioral and cognitive deficits. We divided control and MAM exposed Sprague-Dawley rats into enriched and non-enriched groups and tested performance in the Morris water maze. Another group similarly divided underwent sociability testing and also underwent Magnetic Resonance Imaging (MRI) scans pre and post enrichment. A third group of control and MAM rats without enrichment were trained until they reached criterion on the place avoidance task. MAM rats had impaired performance on spatial tasks and enrichment improved performance of both control and MAM animals. Although MAM rats did not have a deficit in sociability they showed similar improvement with enrichment as controls. MRI revealed a whole brain volume decrease with MAM exposure, and an increase in both MAM and control enriched volumes in comparison to non-enriched animals. In the place avoidance task, MAM rats required approximately 3 times as long to reach criterion as control animals, but with additional training were able to reach control performance. Environmental manipulation and additional training can improve cognition in a rodent MCD model. We therefore suggest that patients with MCD may benefit from appropriate alterations in educational strategies, social interaction and environment. These factors should be considered in therapeutic strategies.

Combined effects of prenatal inhibition of vasculogenesis and neurogenesis on rat brain development.

Malformations of cortical development (MCD) are one of the most common causes of neurological disabilities including autism and epilepsy. To disrupt cortical formation, methylazoxymethanol (MAM) or thalidomide (THAL) has been used to affect neurogenesis or vasculogenesis. Although previous models of MCD have been useful, these models primarily attack a single aspect of cortical development. We hypothesized that simultaneous prenatal exposure to MAM or THAL will lead to the development of a novel and specific type of brain maldevelopment. Rats were prenatally exposed to MAM and THAL. At early postnatal days, brains displayed abnormal ventricular size and hemispheric asymmetry due to altered brain water homeostasis. The postnatal brain was also characterized by gliosis in regions of focal leakage of the blood brain barrier. These morphological abnormalities gradually disappeared at adult stages. Although the adult MAM-THAL rats showed normal cortical morphology, abnormal hippocampal connectivity and mossy fiber sprouting persisted well into adulthood.

The genesis of epileptogenic cerebral heterotopia: clues from experimental models.

The pre-natal administration of methylazoxymethanol acetate (MAM) in rats is able to induce cerebral heterotopia that share striking similarities with those observed in human periventricular nodular heterotopia, a cerebral dysgenesis frequently associated with drug-resistant focal seizures. In the present study, we investigated the mode of neurogenesis in cerebral heterotopia of MAM-treated rats, by analyzing post-natal cytoarchitectural features and time of neurogenesis using bromodeoxyuridine immunocytochemistry. The cytoarchitectural analysis demonstrated the existence, in the early post-natal period, of white matter cellular bands in close anatomical relationship with the heterotopia, which most likely serve as a reservoir of young, migrating neurons for the newly forming heterotopia. The birth dating analysis demonstrated that the period of generation of neurons within the heterotopia and adjacent white matter bands, was extended in comparison to corticogenesis in normal rat brains. In addition, it demonstrated that the heterotopia were formed through a rather precise outside-in (for cortical and periventricular heterotopia) and dorso-ventral (for intra-hippocampal heterotopia) neurogenetic pattern. We hypothesize that the MAM-induced ablation of an early wave of cortical neurons is sufficient to alter per se the migration and differentiation of subsequently generated neurons, which in turn set the base for the formation of the different types of heterotopia. On this basis, we suggest a neurogenetic scheme for MAM-induced heterotopia that can also explain the origin and intrinsic epileptogenicity of periventricular nodular heterotopia in humans.

Dysplastic neocortex and subcortical heterotopias in methylazoxymethanol-treated rats: an intracellular study of identified pyramidal neurones.

Intracellular recordings were obtained using biocytin-filled electrodes from 78 neurones located in both dysplastic neocortex and subcortical heterotopic aggregates in a model of neuronal migration disorder induced in rats by means of a double methylazoxymethanol injection given on embryonic day 15. Both regular spiking and intrinsically bursting pyramidal neurones were found in all of the examined structures and were synaptically activated by subcortical stimulation. In a neuronal subpopulation (22%) located in the neocortex as well as in the subcortical heterotopic aggregates, the injection of depolarising current pulses elicited aberrant firing patterns, consisting of repetitive bursts of APs that gradually increased in duration and eventually merged in a long-lasting discharge. The gradual development of this 'excessive' bursting behaviour suggests a progressive run-down of the slow components of the hyperpolarising afterpotential.

Ubiquitination of apoptotic cells in the developing cerebellum of the rat following ionizing radiation or methylazoxymethanol injection.

Previous studies have shown ubiquitin mRNA induction and protein expression associated with regressive phenomena in some cases of developmentally programmed cell death and experimentally induced apoptosis. Ubiquitin immunoreactivity was examined in the developing cerebellum of the rat following ionizing radiation or methylazoxymethanol (MAM) injection. In irradiated rats, apoptotic cells in the external granule cell layer appeared at 3 h, peaked at 6 h, and decreased thereafter to reach nearly normal values at 48 h. In MAM-treated rats, apoptotic cells in the external granule cell layer were seen at 24 h, peaked at 48 h, and decreased at 72 h. Strong ubiquitin expression was observed in about 15% of apoptotic cells at later stages of apoptosis in both experimental models of induced cell death. In irradiated rats, strong ubiquitin immunoreactivity in apoptotic cells and cellular debris was observed 12 h after irradiation, peaking at 24 h, and decreasing at 48 h. In MAM-treated rats, strong ubiquitin immunoreactivity was found in apoptotic cells and cellular debris at 48 h and decreased at 72 h. Results suggest that activation of the ubiquitin pathway is not a signal that triggers apoptosis but rather a final step in the apoptotic process.

The effect of dietary quercetin and rutin on AOM-induced acute colonic epithelial abnormalities in mice fed a high-fat diet.

Dietary quercetin (QU) and rutin (RU), phenolic flavonoids found in many fruits and vegetables, when fed to mice on a low-fat diet successfully modified the response to azoxymethanol (AOM) by initially inhibiting hyperproliferation and the formation of foci of dysplasia (FADs) and ultimately reducing tumor incidence (Carcinogenesis 12, 1193-1196, 1991). In this study, we tested the efficacy of QU and RU when a high-fat diet was presented. An AIN 76A diet made with 20% corn oil (CO) was supplemented with QU (0.5%, 2.0%, or 5.0%) and RU (2.0% or 4.0%). These five diets, as well as a 5.0% and a 20.0% CO diet, were fed to a group of CF1 female mice for nine weeks. Both QU and RU showed nonsignificant dose-related trends toward normalization of the AOM-induced upward extension of S phase cells. Examination of 500 microns of serially sectioned distal colon revealed that 29% of mice fed the 20% CO control diet were free of FADs. Among the mice fed QU, regardless of dose, > 80% were free of FADs. When the three groups fed QU were pooled and compared with the control 20% CO-fed mice, the degree of protection was significant (p < 0.01). Mice fed RU expressed a level of protection that bordered on the significant (p < 0.08). These data suggest that, regardless of the fat content of the diet, QU and RU are capable of modifying or inhibiting events in the development of chemically induced colonic neoplasia.

The synergistic effect of 1-hydroxyanthraquinone on methylazoxymethanol acetate-induced carcinogenesis in rats.

The synergistic potential of 1-hydroxyanthraquinone (1-HA) on methylazoxymethanol (MAM) acetate-induced carcinogenesis was investigated in rats. A total of 154 inbred ACI/N rats (73 males and 81 females), six weeks old at the start of the experiment, were divided into four groups: group 1 was given i.p. injections of MAM acetate (25 mg/kg body wt), once per week for 2 weeks and then fed the diet containing 1% 1-HA for 42 weeks; group 2 received MAM acetate and was kept on the basal diet alone; group 3 was given 1-HA containing diet alone as for group 1; group 4 was treated as a control. At the termination of the experiment, the carcinogenic effect of MAM acetate and 1-HA in the large bowel or liver exceeded the sum of effects when given alone, indicating that the two chemicals act synergistically in the carcinogenesis of these organs.

Influence of Lactobacillus arabinosus on metabolic enzyme activity of methylazoxymethanol (MAM) acetate in gnotobiotic mice.

The role of Lactobacillus arabinosus in the malignant transformation of tumors of the large intestine was investigated in mice. Methylazoxymethanol (MAM) acetate, at a weekly dose of 0.2 mg/10 g body weight, was given to germfree mice and to mice monocontaminated with either L. arabinosus or Escherichia coli. At sacrifice, the activity of non-specific esterase, beta-glucuronidase, and alcohol dehydrogenase within the liver and intestine was examined biochemically and histochemically. Non-specific esterase activity in the liver and large intestine was significantly higher in L. arabinosus mice than in the other 2 groups. Also, beta-glucuronidase activity in the large intestine and alcohol dehydrogenase activity in the liver were significantly greater in L. arabinosus mice than in the other groups. Esterase was localized in the mitochondria and absorptive granules within the mucosal epithelium of the large intestine. An apparent increase in the number of certain organelles was observed in the L. arabinosus mice, compared with the other groups. These results suggest that L. arabinosus plays an important role in MAM acetate tumorigenesis and malignant transformation.

[Emulsifiers which reduce the latency of the development of colonic cancer--sodium lauryl sulfate and methylazoxymethanol].

The effect of emulsifier (nonionic surfactant) on the production of adenocarcinoma by methylazoxymethanol acetate in the large intestine of rats was studied. Following emulsifier, sodium lauryl sulfate administration, many cases of undifferentiated adenocarcinoma consisting of anaplastic glandular cells were induced in the experimental groups. Lymphatic invasion by cancer cells was found in 3 cases and metastasized to other organs in 6 cases. On the contrary, the control group (administered methylazoxymethanol acetate only) revealed well-differentiated adenocarcinoma in many cases. This fact may be due to an emulsifier used as a vehicle for the chemical, and the emulsifier might activate the character of promotion to carcinogenisity as a secondary agent. By virtue of the strong penetrating property of the emulsifier, colonial carcinogenesis seems to be enhanced.

Tumors of the anal region induced in mice painted with methylazoxymethanol acetate.

The carcinogenicity of methylazoxymethanol acetate (MAM acetate) was examined in 91 BALB/c mice by painting it on the anal region. Carcinomas and adenomas of the perianal sebaceous gland were induced in 23 of 24 male (96%) and 16 of 30 female (53%) mice and keratoacanthoma developed in one of 24 male mice within 30 weeks after treatment with MAM acetate. Vascular tumors of the liver and fat issue of the abdominal cavity also developed in 16 of 24 male (67%) and 3 of 30 female (10%) mice treated with this drug. Microscopic adenocarcinomas were found in the rectal mucosa of 3 of 24 male mice adjacent to the anorectal junction. The sex difference in the incidence of tumors is briefly discussed.

Leiomyomas and leiomyosarcomas of the kidney in 4-day-old BUF rats given methylazoxymethanol acetate intraperitoneally.

The effect of age and sex on the development of renal tumors was studied in inbred BUF male and female rats 4 days, 5, 8, 12, 24, or 52 weeks old. Methylazoxymethanol (MAM) acetate was injected ip (20 mg/kg body wt) once weekly for 9 weeks. Animals 52 weeks old died from hepatic and/or renal necrosis; however, animals of other ages survived 24-42 weeks. Female rats 4 days old were susceptible to the development of leiomyomas and leiomyosarcomas of the kidney, whereas 4-day-old male rats had a few leiomyomas. Adenomas and carcinomas of the kidney and nephroblastomas were not observed. It was concluded that the aglycone of cycasin, MAM, is important in the induction of leiomyosarcomas of the kidneys in 4-day-old rats.

Carcinomas of the colon in Buffalo strain rats given intraperitoneal injections of methylazoxymethanol acetate.

Buffalo strain male and female rats were given methylazoxymethanol acetate (20% solution) intraperitoneally 20 mg/kg body weight weekly for 9 weeks, and killed 27 weeks later. Carcinomas of the large intestine, predominantly in the descending colon, developed in some of the rats. The incidence was higher in male than in female rats. Polypoid carcinomas were observed more often in male rats and sessile carcinomas with metastases in the female rats. Carcinomas were not observed in organs other than the large intestine.