Effect of streptozotocin induced diabetes on rat hippocampus.

OBJECTIVE: The present study was designed to evaluate the diabetes induced neuronal damage in the hippocampus of the rat brain. BACKGROUND: Diabetes mellitus is an endocrine metabolic disorder of impaired carbohydrate, fat and protein metabolisms characterized by chronic hyperglycemia. The neurological consequences of diabetes mellitus in the brain have gained attention most recently. MATERIALS AND METHODS: Male albino rats of Wistar strain, aged 30 days were used. The rats were divided into (A) Normal Control (B) Vehicle Control (C) 15 days of Streptozotocin (STZ), (D) 30 days of STZ, (E) 45 days of STZ, (F) 60 days of STZ diabetic groups (n = 6 in each group). Blood glucose levels and body weight were measured before STZ injection, 2 days after STZ injection and on the day of sacrifice. At the end of the experimental period rats were scarified and brains were processed for cresyl violet staining and the number of survived neurons in the hippocampus was quantified. RESULTS: Microscopic examination of cresyl violet stained sections of diabetic rat hippocampus showed significant and reliable changes. There was a significant difference in the number of survived neurons especially in 30 days of STZ, 45 days of STZ and 60 days of STZ diabetic groups compared to normal control group. CONCLUSION: The results of our study indicated that diabetic complications can cause rapid damage to the neurons in the hippocampus (Fig. 12, Ref. 22).

Anti-inflammatory effect of Inonotus obliquus, Polygala senega L., and Viburnum trilobum in a cell screening assay.

AIM OF THE STUDY: The purpose of the study was to assess the anti-inflammatory effects of the mushroom Inonotus obliquus (Chaga), Polygala senega (Senega) and Viburnum trilobum (Cranberry) bark extract fractions from locally produced materials in lipopolysaccharide (LPS) induced murine macrophage RAW 164.7 cells. MATERIALS AND METHODS: Four fractions from each of the three extracts were obtained: (80% ethanol extracted; Fa), (water-soluble polysaccharide fraction; Fb), (Polyphenolic fraction; Fc) and (ETOAc/H(2)O extracted fraction; Fd). These extract fractions were tested in the cell screening system at 50,100 and 500 microg/ml for their ability to inhibit LPS induced inflammatory cytokines IL-1beta, TNFalpha and IL-6. Supernatants from LPS alone treated cells were used as control. The cytokines in the cell culture supernatants following treatments with extract fractions were quantified by ELISA method, using 96 well ELISA plates. RESULTS: All fractions of the extracts significantly inhibited (p<0.05) the levels of IL-1beta, IL-6 and TNFalpha except the polyphenolic Fc fraction of Senega which showed an increased production of IL-6. Furthermore, each fraction showed a dose-dependant anti-inflammatory effect. Nitric oxide production was not affected by cranberry and senega, while Chaga significantly reduced NO production in murine macrophage cell assay. CONCLUSIONS: These results demonstrate that the extracts obtained from the root of Polygala senega L., bark of Viburnum trilobum, and the mushroom Inonotus obliquus possess anti-inflammatory properties when tested in a RAW 264.7 macrophage cell system.

Clonal chromosomal changes in chronic lymphocytic leukemia.

Cytogenetic analysis was carried out in 28 B-CLL patients (21 males and 7 females, 38-85 years old, with median age 64 years, disease stage O-IV). Peripheral nominator cells (1 x 10(7)) or isolated B-lymphocytes were incubated in vitro for 5-7 days. The cells were stimulated by pokeweed mitogen (PWM), or phorbol myristate-acetate (PMA), with or without 10% conditioned medium (CM) derived from a T cell leukemia line or 10% B-cell growth factor (BCGF). Twenty-two patients (79%) responded to PWM + CM; 5 out of 5 patients responded to PWM + BCGF. The average mitotic index (+/- S.E.M.) for PWM, PMA, PWM + CM, PMA + CM, PWM + BCGF were 0.13 +/- 0.01, 0.24 +/- 0.13, 0.51 +/- 0.11, 0.14 +/- 0.06 and 0.63 +/- 0.15, respectively. Cytogenetic analysis revealed the presence of abnormal karyotypes in 22 patients. Fourteen patients (50%) had clonal chromosome aberrations which included: monosomy 1, 9, 17, 18, 21, and X chromosome, and trisomy of chromosomes 7, 9, 20, 21 and 22. The clonal structural aberrations were i(6q), inv(12) (q15q24), del(5) (p13p15), del(10) (q24). No homogeneously staining regions (HSR) were observed. Four patients with resistance to anti-neoplastic drugs showed the presence of double minute chromosomes (dmin) ranging in frequency from 5 to 50%.

Reduced expression of aphidicolin-induced common fragile sites in peripheral lymphocyte chromosomes of patients with B-cell chronic lymphocytic leukemia.

The occurrence and frequency of aphidicolin-induced common chromosomal fragile sites were examined in 12 B-cell chronic lymphocytic leukemia (B-CLL) patients (ten males and two females) and three normal individuals. The mononuclear cells separated by Ficoll-Hypaque gradient were cultured in vitro for 96 hours stimulated by pokeweed mitogen (PWM) in combination with T-leukemia cell conditioned medium or 10% B-cell growth factor. For the final 24 hours the cells were treated with aphidicolin (0.07 microgram/ml). Results indicate that there was a significant reduction in the overall mean frequency of common fragile sites in CLL patients with a wide individual variation. Fragile sites were found to be localized either on a single chromatid or both chromatids, but rarely involved homologous chromosomes. No definite relationship between the frequency of fragile sites and the staging of CLL disease was observed. A significant reduction and variability in the frequency of fragile sites suggest the heterogenous nature of B-CLL and probably a different mechanism of induction of fragile sites in CLL cells compared to controls.

Maternal and fetal chromosomal aberrations in mice following prenatal exposure to subembryotoxic doses of lead nitrate.

Maternal exposure to low levels of lead nitrate (12.5, 25, 50 mg/kg body weight), administered on the 9th day of gestation, did not cause embryonic resorption and fetal lethality, but induced chromosomal deletions and other forms of aberrations in fetal liver and maternal bone marrow cells.

Relationship of embryotoxicity to genotoxicity of lead nitrate in mice.

Frequency of sister chromatid exchanges (SCEs), nucleolar organizing regions (NORs) and chromosomal aberrations were analysed, in maternal bone marrow and fetal liver and/or lung cells of ICR Swiss Webster mice, following maternal exposure to lead nitrate on gestational day 9. The number of implantations and morphological changes in day 18 fetuses, following the treatment, were also noted. Chemical analysis of lead in maternal and fetal tissues showed that it is readily transferred across the placenta. Lead caused a moderate, but statistically significant, increase in the frequency of SCEs in maternal bone marrow cells and significant reduction in NORs at the 2 highest dose levels (150 and 200 mg/kg b.w.). Lead treated animals showed several specific chromosomal aberrations, mostly deletions in maternal bone marrow and fetal cells. Aneuploidy was found to be frequently associated with the lowest dose levels of lead nitrate (100 mg/kg). Maternal treatment with lead nitrate also significantly increased embryonic resorptions and reduced placental weights. The results suggest that the embryotoxic effects of lead might be associated with the chromosomal changes.

Embryotoxicity and in vivo cytogenetic changes following maternal exposure to cadmium chloride in mice.

Cadmium is a well-known teratogen in laboratory animals and a widespread environmental pollutant. The frequencies of sister chromatid exchanges (SCEs), nucleolar organizing regions (NORs) and chromosomal aberrations were analysed in maternal bone marrow and fetal liver and/or lung cells of mice, following maternal treatment with cadmium chloride, on gestational days 8 through 10. The embryotoxic effects and morphological changes on day 18 fetuses were also studied. Cadmium chloride is readily transferred across the placenta and significant levels were detected in both the placenta and fetus. No significant changes in the frequencies of SCEs or NORs in maternal and fetal cells were observed following exposure to cadmium chloride. Fetal tissues showed mitotic inhibition at the highest dose levels (8.4 and 11.4 mg/kg, b.w.). Maternal treatment with cadmium chloride increased embryonic resorptions and fetal lethality, as well as reduced placental weight; however, it did not produce significant chromosomal changes except at the highest dose level (11.4 mg/kg).

Cytogenetic effects of vaginally administered gossypol in murine bone marrow cells.

The cytogenetic effects of gossypol were evaluated by determining the frequency of sister chromatid exchanges (SCEs), percentage of pulverized metaphases, mitotic indices and micronuclei in bone marrow cells of mice treated per vaginam. A dose-dependent increase in the frequency of SCEs was observed when gossypol suspended in corn oil was administered at dosages of 10, 20 or 40 micrograms/g. In comparison with controls, incidences of SCEs were significantly higher in mice given 20 and 40 micrograms/g gossypol, whereas the mitotic indices, percentages of pulverized metaphases and the frequency of interphase micronuclei in treated animals were not different from their control counterparts. The SCE data suggest that gossypol has a DNA-damaging potency in murine bone marrow cells.

Induction of sister chromatid exchanges and chromosome damage by gossypol in bone marrow cells of mice.

The chromosome-damaging potential of gossypol was evaluated by scoring sister chromatid exchanges (SCEs), determining the percentage of pulverized metaphases and the mitotic index in bone marrow cells of mice. Bone marrow cells were collected approximately 21 hours after the intraperitoneal (0,20,40,80, or 160 micrograms/g) and oral (0,40,80, or 160 micrograms/g) administration of gossypol acetic acid. Irrespective of the dosing schedule (single or multiple doses), the vehicle used (physiological saline, corn oil, or 10% aqueous ethanol), and the route of administration, the mean SCE count per cell was significantly higher (P less than 0.05) in gossypol-treated groups than their control counterparts. At 80 and 160 micrograms/g dose levels, the occurrence of metaphase chromosome pulverization was significantly greater, while mitotic index values were markedly lower than those of the corresponding control values. The results suggest that gossypol is a potentially mutagenic and clastogenic agent in murine bone marrow cells.

Environmental monitoring for genotoxicity: in vivo sister chromatid exchange in the house mouse (Mus musculus).

Sister chromatid exchange (SCE) values were determined in bone marrow cells isolated from mouse (Mus musculus) femurs after injections of 5-bromo-2'-deoxyuridine (BrdU) and 5-fluorodeoxyuridine (FrdU). Male mice of C3H/J, C57BL/6J, and DBA/2 strains maintained in the laboratory gave mean SCE values of 3.42 +/- 0.07, 3.62 +/- 0.08, and 3.97 +/- 0.13, respectively. Males obtained from natural populations of southwestern Ontario had a higher mean SCE value (6.02 +/- 0.16), as did inbred males maintained in outdoor enclosures for at least 3 weeks (5.07 +/- 0.22). Wild mice housed in the laboratory for 9 months or longer had SCE values similar to laboratory bred mice (3.46 +/- 0.05). The SCE values in wild-caught mice were inversely proportional (r = -0.49) to the distance between the sites where these animals were collected and the nearest major industrial center. Based on these results, SCE analysis in mice is proposed as a possible first-line monitoring procedure for the detection of general changes in environmental genotoxicity.

Fluorodeoxyuridine-induced sex differences in baseline sister-chromatid exchanges in C57BL/6 mice and Chinese hamsters.

The baseline sister-chromatid exchanges (SCEs) and the percentage of first (M1), second (M2) and third or higher metaphase (M3+) chromosomes were analysed in bone-marrow cells of male and female C57BL/6 mice and Chinese hamsters following serial intraperitoneal injections of 40 micrograms/g body weight (b.w.) of 5-bromo-2'-deoxyuridine (BrdUrd) and 2 micrograms/g b.w. of 5-fluorodeoxyuridine (FdUrd) or 40 micrograms/g b.w. of BrdUrd and 10 micrograms/g b.w. of deoxycytidine (dC). Female animals receiving BrdUrd/FdUrd showed significantly higher (P less than 0.01) baseline SCEs compared to the other groups. No sex difference in the baseline SCEs was found in animals treated with BrdUrd/dC. The distribution patterns of M1, M2 and M3+ metaphases in BrdUrd/FdUrd-treated animals differ significantly from those in BrdUrd/dC-treated animals.

Lack of induction of sister chromatid exchanges and of mutation to 6-thioguanine resistance in V79 cells by butylated hydroxyanisole with and without activation by rat or hamster hepatocytes.

The antioxidant butylated hydroxyanisole (BHA) and the promutagen/carcinogen 7,12-dimethylbenz[a] anthracene (DMBA) were examined for mutagenicity and the induction of sister chromatid exchanges (SCE) in a hepatocyte-mediated mutation assay with V79 Chinese hamster lung cells. Rat and hamster hepatocytes, prepared by in situ collagenase perfusion, were compared in the mutation assay to determine whether there are species differences in the ability to activate BHA and DMBA to ultimate mutagens. At the marginally cytotoxic concentration of 1.0 microM (2.6 micrograms/ml), DMBA induced a significant increase in the frequency of SCE and in the number of mutations to 6-thioguanine resistance (6-TGR) at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus with either rat or hamster hepatocyte-mediated activation, but induced highest mutation frequencies with rat hepatocytes. These findings support the contention that species differences can affect mutational response in hepatocyte-mediated assays with V79 cells. BHA was strongly cytotoxic to V79 cells at dose levels in excess of 0.3 mM (54 micrograms/ml). In contrast to DMBA, BHA showed no evidence of genotoxicity at marginally cytotoxic concentrations up to and including 0.3 mM as shown by the inability of this antioxidant to increase the frequency of sister chromatid exchanges or to induce mutations to 6-thioguanine resistance when activation was provided by rat or hamster hepatocytes.

In vitro short-term testing systems as prescreens for potential carcinogens: simultaneous detection of sister chromatid exchanges and mutation response in a cell-mediated assay with V79 cells.

Sister chromatid exchange (SCE) is recognized as a sensitive indicator of genetic damage, and this has led numerous investigators to suggest that the analysis of SCE can provide a useful step toward the identification of environmental mutagens and/or carcinogens. To explore this approach, we measured SCE induction in V79 Chinese hamster lung cells and the frequency of mutation to 6-thioguanine resistance at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) locus in a cell-mediated mutation assay. Karyotypic analysis of V79 cells showed a stable modal chromosome number of 22 and an XY chromosome complement. When exposed to the procarcinogen 7,12-dimethylbenz[a]anthracene (DMBA) at marginally cytotoxic dose levels of 1.0, 0.5 and 0.25 microM (2.6, 1.3 and 0.65 micrograms/ml), SCE frequencies were highest within the first 24 h of activation with rat or hamster hepatocytes, showed somewhat lower values after 48 h of activation, and, following withdrawal of the chemical, declined to background levels during the period of expression. While this decline may involve several factors, the possibility is not excluded that DNA repair can contribute to the progressive elimination of SCE. The induction of SCE in V79 cells appeared unrelated to the expression of single-point mutation at the HGPRT locus. These findings demonstrate the advantage of multiple endpoint analysis which enabled cytotoxicity, mutagenicity and conditions optimal for the induction of SCE to be determined concurrently in a hepatocyte-mediated assay with V79 cells.