Influence of teniposide (VM-26) on radiation-induced damage to mouse spermatogenesis: a flow cytometric evaluation

The effect of teniposide (VM-26) 0.05 mg/kg body weight treatment on spermatogenesis of mice exposed to 0, 0.5, 1, 2, and 3 Gy gamma-radiation was evaluated flow cytometrically. Whole body irradiation with 1 to 3 Gy resulted in a significant decline in testis weight from Day 14 to 35 post-irradiation depending on the exposure dose. Treatment of mice with teniposide before irradiation advanced the decline in testicular weight by several days, especially at 3 Gy, where a significant decline in testicular weight was observed at Day 7 post-irradiation when compared with the double distilled water (DDW)+irradiation group. The relative percentage of the 2C population declined significantly in the VM-26+irradiation group in comparison with the DDW+irradiation group at various post-irradiation time periods depending on the exposure dose. A significant depletion in the relative percentage of S-phase cells was observed as early as Day 1 post-irradiation in the VM-26+irradiation group when compared with the DDW+irradiation group after exposure to 1 to 3 Gy. This decline continued up to Day 21 post-irradiation after exposure to 2 Gy. The relative percentage of primary spermatocytes showed a consistent decline after exposure to various doses of gamma-radiation in the VM-26+irradiation group when compared with the DDW+irradiation group at different time periods, with a few exceptions, especially at higher doses. The pattern of decline in the relative percentage of round spermatids was similar to that of primary spermatocytes, where a significant decline was observed at various post-irradiation time periods in the VM-26+irradiation group in comparison with the DDW+irradiation group. These changes in the relative germ cell percentages are manifested as alterations in the ratios of various germ cell populations. The 4C:2C ratio declined consistently from Day 1 to Day 70 post-irradiation in the VM-26+irradiation group at all exposure doses. Similarly, the 4C:S-phase ratio in the VM-26+irradiation group also showed a significant decline at different post-irradiation time periods when compared with the DDW+irradiation group depending on the exposure dose. The reduction observed in the relative percentages of various cell populations was higher in the combination group when compared with the DDW+irradiation controls, indicating potentiation of damage to male germ cells by teniposide treatment before irradiation.     

Effect of teniposide (VM-26) on the cell survival, micronuclei-induction and lactate dehydrogenase activity on V79 cells

The genotoxic effect of 0, 1, 5, 10, 25, 50 and 100 nM teniposide (VM-26) treatment was studied on cultured V79 cells. Treatment of V79 cells with different concentrations of teniposide resulted in a concentration-dependent decline in the cell survival and growth kinetics. VM-26 treatment also caused alteration in the cell proliferation kinetics as evidenced by the increase in the frequency of mononucleate cells, with a consequent decline in the frequency of binucleate cells in a concentration-dependent manner at all the post-treatment time periods. Exposure of V79 cells to different concentrations of VM-26 resulted in a concentration related elevation in the frequency of micronucleated binucleate (MN) cells. The frequency of MN was significantly higher in VM-26 treated cells than that of non-drug treated cells at all the post-treatment time periods. A peak frequency of MN was observed at 16 h post-treatment that declined thereafter. The release of lactate dehydrogenase increased with the increase in drug concentration and a maximum LDH release was observed at 0.5 h post-treatment after exposure to 10-100 nM VM-26. While a peak value was observed at 1 and 2 h for 5 and 1 nM VM-26, respectively. The biological response was evaluated by determining the relationship between micronuclei and cell survival. The cell survival declined with increasing MN frequency, resulting in a close but an inverse relationship between the cell survival and micronuclei-induction. The dose effect relationships for micronuclei induction, LDH release and biological response was linear quadratic.     

Evaluation of teniposide (VM-26)-induced toxicity on mouse spermatogenesis by flow cytometry

Alteration in the testicular weight and various germ cell populations was studied in male mice treated with different doses (0.05, 0.25, 0.5, 1.0 and 2.0 mg/kg b. wt.) of teniposide (VM-26) at various post-treatment time periods. Treatment of mice with different doses of teniposide did not significantly alter the testicular weights, irrespective of the drug dose used. Flow-cytometric analysis of germ cells of the untreated control mice testes revealed four distinct DNA peaks corresponding to elongated spermatids (HC), round spermatids (1C), spermatogonia and non-germ cells (2C) and primary spermatocytes (4C). The region between 2C and 4C peaks represents cells that are actively synthesizing DNA (S-phase cells). Treatment of mice with different doses of teniposide resulted in a significant depletion in the relative percentage of spermatogonia from day 2 to 35 post-treatment depending on the drug dose. DNA-synthesizing, i.e. S-phase, cells declined significantly at day 1 post-treatment and continued to decline up to day 70 post-treatment for all the drug doses studied, except 2 mg/kg drug dose at day 28 post-treatment. A significant decline in the relative percentage of primary spermatocytes (4C) was observed at day 7 that continued up to day 70 post-treatment depending on the drug dose. Round spermatids (1C) declined significantly at day 21 post-treatment after administration of 0.25--2.0 mg/kg VM-26. The relative percentage of elongated spermatids showed a significant decline at day 28 after 1 and 2 mg/kg drug treatment. These alterations in different germ-cell populations are reflected in the various germ-cell ratios. The 4C:2C ratio showed a significant decline at day 7 and 14 post-treatment after 1 and 2 mg/kg VM-26 treatment, while the 1C:2C ratio declined significantly at day 21 post-treatment in the mice treated with 0.5 and 2.0 mg/kg of VM-26. 4C:S-phase and 1C:4C ratios increased significantly from day 1 to 70 post-treatment, depending on the drug dose. Our study demonstrates that the treatment of mice with low doses of VM-26 exerts cytotoxic effects on various germ-cell populations.     

Correlation between cell survival, micronuclei-induction, and LDH activity in V79 cells treated with teniposide (VM-26) before exposure to different doses of gamma radiation.

The influence of teniposide (VM-26) treatment was studied on the radiation-induced alterations in cell survival, micronuclei (MN) formation and lactate dehydrogenase (LDH) release in V79 cells. Treatment of V79 cells with 10 nM teniposide before exposure to different doses of gamma radiation resulted in a significant decline in the cell survival when compared with the PBS + irradiation group. The decline in cell survival was dose related. The cell proliferation indices also declined in a dose-dependent manner in both PBS + irradiation and VM-26 + irradiation groups. The decline was higher in the VM-26 + irradiation group in comparison with the PBS + irradiation group. In contrast, the frequency of micronuclei increased in a dose-related manner in both PBS + irradiation and VM-26 + irradiation groups. However, the frequency of micronuclei was significantly greater in the latter group when compared with the former group at all the post-irradiation time periods studied. The LDH contents increased in a dose-dependent manner in both PBS + irradiation and VM-26 + irradiation groups at all the post-irradiation time periods evaluated. This elevation in LDH contents was significantly greater in the VM-26 + irradiation group in comparison with the PBS + irradiation group.     

Inhibition of radiation-induced clastogenicity by Aegle marmelos (L.) correa in mice bone marrow exposed to different doses of gamma-radiation

The frequency of micronucleated polychromatic (MPCE), normochromatic erythrocytes (MNCE), and polychromatic/normochromatic erythrocyte ratio (PCE/NCE), was studied in the bone marrow of mice orally administered with 0, 200, 225, 250, 275 and 300 mg/kg body weight of hydroalcoholic leaf extract of Aegle marmelos (AME). Treatment of mice with AME, once daily for 5 consecutive days, before exposure to 2 Gy resulted in a significant decline in the frequency of MPCE when compared to the non-drug-treated irradiated control. The greatest reduction in MPCE was observed for 250 mg/kg body weight AME, accompanied by the highest polychromatic erythrocyte to normochromatic erythrocyte ratio, in comparison with the non-drug-treated irradiated control. Therefore, further studies were carried out using this dose of AME, where the animals were administered with 250 mg/kg body weight of AME before exposure to 0, 0.5, 1, 2, 3 and 4 Gy of gamma-radiation and evaluated at 12, 24, 36 and 48 hours post-irradiation. Whole body irradiation of mice to different doses of gamma-radiation resulted in a dose-dependent increase in the frequency of MPCE at all post-irradiation times. Treatment of 250 mg/kg AME orally (p.o.) before irradiation significantly reduced the frequency of MPCE at all post-treatment times. The frequency of MPCE increased with time, reached a peak level at 24 hours, and declined thereafter. The occurrence of MNCE has also shown a pattern similar to MPCE, except that the MNCE frequency reached a peak level by 48 hours. The AME significantly reduced the frequency of MNCE at all post-irradiation times, when compared to the non-drug-treated irradiated group. Treatment of mice with AME before exposure to different doses of gamma-radiation resulted in the inhibition of a radiation-induced decline in the PCE/NCE ratio, when compared with the concurrent irradiated controls. To gain insight into the mechanism of action, AME was tested for its antioxidant effects in cell-free chemical systems using H2O2/FeSO4 to generate hydroxyl (*OH) radicals, which were measured by a fluorescent probe, 2V, 7V-dichlorofluorescin diacetate (DCFH/DA). Xanthine/xanthine oxidase was used to generate superoxide (O2*-) anion radical, which was measured by a fluorescent probe dihydroethidium (DHE). AME significantly reduced fluorescence in a concentration dependent manner, indicating its efficacy to scavenge free radicals. Our results demonstrate that one of the mechanism of reduction in the radiation-induced DNA damage in mice bone marrow by AME may be due to scavenging of free radicals and elevation in the antioxidant status, as previously reported.     

Melatonin reduces paraquat-induced genotoxicity in mice

The protection afforded by melatonin against paraquat-induced genotoxicity in both bone marrow and peripheral blood cells of mice was tested using micronuclei as an index of induced chromosomal damage. Melatonin (2 mg/kg) or an equal volume of saline was injected i.p. into mice 30 min prior to the i.p. administration of paraquat (two injections of 15 mg/kg; the paraquat injections were given with a 24 h interval) and thereafter at 6 h intervals to the conclusion of the study (72 h). Using fluorescence microscopy, the number of micronuclei in polychromatic erythrocytes (MN-PCE) per 2000 PCE (1000 PCE/slide) per mouse was counted both in blood and bone marrow, and the ratio of PCE to normochromatic erythrocytes (NCE) (PCE/NCE) was calculated. Paraquat treatment increased the number of MN-PCE at 24, 48, and 72 h, both in peripheral blood and bone marrow cells, while no differences were observed in the PCE/NCE ratio. Melatonin inhibited the paraquat-induced increase in MN-PCE by more than 50% at 48 and 72h. Paraquat toxicity is believed to be due to free radical generation. Since melatonin is known to be an efficient free radical scavenger, it is concluded that melatonin's protection against paraquat-induced genotoxicity is mediated, at least in part, by its free radical scavenging activity.     

Increased micronucleus frequency in rat bone marrow after acrylamide treatment

This study was performed to investigate whether acrylamide (AA), occured during cooking carbohydrate-rich foods at high temperature, increased the frequency of micronucleated polychromatic erythrocytes (MNPCEs) in rat bone marrow. For this purpose AA, dissolved in distilled water, was administered to 8-week old male Sprague Dawley rats at single oral doses of 0, 125, 150 or 175 mg/kg b.w. After 48 h from AA treatment, the bone marrow samples were analysed for the frequency of MNPCEs. The cytotoxic effect of AA on bone marrow was also tested by assessing polychromatic erythrocyte/normochromatic erythrocyte (PCE/NCE) ratio. It was found that all three doses applied significantly increased the frequency of MNPCEs and this increase was 3.75-fold in rats given the highest administered dose of AA. In addition AA decreased the PCE/NCE ratio, which is indicative of bone marrow cytotoxicity when compared to the control group. This study displayed that AA increased the formation of micronuclei in polychromatic erythrocytes (PCEs) of rat bone marrow and this increase might have resulted from administrating the high dose level of AA to rats by gavage instead of by i.p. injection.     

Assessment of acetamiprid-induced genotoxic effects in bone marrow cells of Swiss albino male mice

Acetamiprid (ACE), a neonicotinoid insecticide, is widely used in agriculture either alone or in combination with other insecticides. A combined approach employing micronucleus test (MNT) and chromosomal aberrations (CA) assay was utilized to assess the genotoxic effects of ACE in bone marrow of Swiss albino male mice. Acetamiprid was administered i.p. daily at 4.6 and 2.3?mg/kg/day along with 3% gum acacia as negative control for 60 and 90?days and cyclophosphamide (50?mg/kg b.wt.) as positive control. ACE treatment resulted in a dose-dependent increase in the frequencies of micronuclei per cell and chromosomal aberrations in bone marrow cells. The increased micronuclei formation in total erythrocyte cells (immature PCEs and mature NCEs) was observed only at higher dose level (4.6?mg/kg b.wt.) administered for 90?days. The test also indicated the cytotoxic effect of higher dose level of pesticide by PCE/NCE ratio. The number of chromosomal aberrations were increased in the pesticide treated group compared to the negative control group, although significant increase was observed only in the group exposed to higher dose level of pesticide for both 60 and 90?days. Thus, daily exposure of ACE at a dose level of 4.6?mg/kg body weight for 60 and 90?days caused genotoxic and cytotoxic effects on the somatic cells of Swiss albino male mice.     

Chemoprotective effects of hesperidin against genotoxicity induced by cyclophosphamide in mice bone marrow cells

The preventive effect of hesperidin as a flavonoid was investigated in mouse bone marrow cells against genotoxicty induced by cyclophosphamide. Mice were orally (gavages) pretreated with solutions of hesperidin at four different doses (50, 100, 200, and 400 mg/kg b.w.) for five consecutive days. Mice were injected intraperitoneally on the fifth day with cyclophosphamide (50 mg/kg b.w.) and killed after 24 h for the evaluation of micronucleated polychromatic erythrocytes (MnPCEs) and the ratio of PCE/(PCE+NCE) (polychromatic erythrocyte/ polychromatic erythrocyte + normochromatic erythrocyte). Three last doses of hesperidin significantly reduced frequency of MnPCEs induced by cyclophosphamide (p<0.0001). Hesperdin at dose 200 mg/kg b.w. reduced MnPCEs 2.37 time and also completely normalized PCE/ (PCE+NCE) ratio. Histological examination of bone marrow showed that hesperidin affected on proliferation and hyper cellularity of immature myeloid elements in bone marrow that reduced by cyclophsopahmide. It is obvious that hesperidin, may with antioxidative activity, reduced the oxidative stress and genotoxicity induced by cyclophosphamide in mouse bone marrow cells.     

In vivo genotoxicity assessment of nerolidol

Nerolidol is a sesquiterpenoid component of essential oil used as a flavor and aroma enhancer. It has also been studied as a topical skin penetration enhancer, and has inhibitory activities against S. aureus and E. coli, among other activities. The objective of this study was to evaluate the ability of a single nerolidol treatment to induce DNA damage in peripheral blood and liver cells of mice and micronuclei in polychromatic erythrocytes of bone marrow cells of the same animals. In the dose range‐finding assays, the maximum tolerated dose was higher than 2000 mg kg^?1. The doses used in the experiments were 250, 500 and 2000 mg kg^?1, administered by gavage in a single dose. Peripheral blood cells were collected 4 and 24 h after the treatments and liver cells 24 h after. At least 100 nucleoids per cell type/animal were analyzed to determine the DNA damage scores and 2000 PCEs per animal for micronuclei in PCEs. The positive control was N‐nitroso‐N‐ethylurea 50 mg kg^?1. Cytotoxicity was assessed by scoring 200 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE:NCE ratio). The results showed that nerolidol induced weak levels of dose‐related DNA damage in both types of cells analyzed, and enhanced the average number of micronucleated cells in the two high doses tested. The PCE:NCE ratio showed no cytotoxicity for the three doses of the compound. The data obtained support the view that nerolidol induces clastogenicity and very weak genotoxicity in the mouse cells tested. Copyright © 2010 John Wiley & Sons, Ltd.     

Protective effect of hawthorn extract against genotoxicity induced by cyclophosphamide in mouse bone marrow cells

The preventive effect of hawthorn (Crataegus microphylla) fruit extract was investigated in mouse bone marrow cells against genotoxicity induced by cyclophosphamide. Mice were orally (gavages) pretreated with solutions of hawthorn extract which was prepared at five different doses (25, 50, 100, 200 and 400mg/kg b.w.) for seven consecutive days. Mice were injected intraperitoneally on the seventh day with cyclophosphamide (50mg/kg b.w.) and killed after 24h for the evaluation of micronucleated polychromatic erythrocytes (MnPCEs) and the ratio of PCE/(PCE+NCE) (polychromatic erythrocyte/polychromatic erythrocyte+normochromatic erythrocyte). All of five doses of extract significantly reduced MnPCEs induced by cyclophosphamide (P<0.0001). Hawthorn extract at dose 100mg/kg b.w. reduced MnPCEs 2.5 time and also completely normalized PCE/(PCE+NCE) ratio. Hawthorn extract exhibited concentration-dependent antioxidant activity on 1,1-diphenyl-2-picryl hydrazyl free radical. Hawthorn contains high amounts of phenolic compounds; the HPLC analysis showed that it contained chlorogenic acid, epicatechin and hyperoside. It is obvious that hawthorn, particularly flavonoids constituents with antioxidative activity, reduced the oxidative stress and genotoxicity induced by cyclophosphamide in mouse bone marrow cells.     

Lack of in vivo clastogenic activity of grape seed and grape skin extracts in a mouse micronucleus assay.

Meganatural brand grape seed extract (GSE) and grape skin extract (GSKE), containing proanthocyanidin polyphenolic compounds, are intended for use in food as functional ingredients exhibiting antioxidant activity. Proanthocyanidins, as well as the minor constituent phenolic compounds in GSE and GSKE, are present naturally in many foods such as fruits, vegetables, chocolate, tea, etc., and on average people consume 460-1000 mg/day of these combined substances. While some polyphenolic compounds, tested individually, have demonstrated antitumorigenic or antipromotional activity, at least one minor component of GSE and GSKE, quercitin, has exhibited positive activity in Salmonella and other in vitro mutagenicity assays. As part of a program to investigate the safety of GSE and GSKE, these products were tested for in vivo clastogenic activity and/or disruption of the mitotic apparatus by detecting micronuclei in polychromatic erythrocyte (PCE) cells in Crl:CD-1(ICR) BR mouse bone marrow. The appropriate test article was dissolved in 0.5% carboxymethylcellulose and dosed by oral gavage to five males/test article/dose level/harvest time point. Animals were dosed at 500, 1000 and 2000 mg/kg. Five animals dosed with either test article at 500, 1000 and 2000 mg/kg dose levels and five animals dosed with the cyclophosphamide (80 mg/kg) positive control were euthanized approximately 24 h after dosing for extraction of bone marrow. Five animals dosed with either test article at the 2000 mg/kg dose level and five animals dosed with the vehicle control article were euthanized approximately 24 and 48 h after dosing for extraction of bone marrow. At least 2000 PCEs per animal were analyzed for frequency of micronuclei. Cytotoxicity was assessed by scoring the number of PCEs and normochromatic erythrocytes (NCEs) in at least the first 500 erythrocytes for each animal. For both GSE and GSKE, no statistically significant increase in micronucleated PCEs was observed at any dose level or harvest time point. GSE produced indication of cytotoxicity (decreased PCE:NCE ratio) at the 2000 mg/kg dose level for the 48-h harvest time point, confirming that the test article reached the target bone marrow in significant amount. Meganatural GSE and Meganatural GSKE were evaluated as negative in the mouse bone marrow micronucleus assay under the conditions of this assay.     

Genotoxicity studies on licorice flavonoid oil (LFO)

Licorice flavonoid oil (LFO) is a new functional food ingredient. In this study, the genotoxicity of LFO was investigated using a test battery of three different methods. In a reverse mutation assay using four Salmonella typhimurium strains and Escherichia coli, LFO did not increase the number of revertant colonies in any tester strain with or without metabolic activation by rat liver S9 mix. In a chromosomal aberration test using Chinese hamster lung (CHL/IU) cells, LFO did not induce any chromosomal aberrations either in the short period test without rat liver S9 mix or in the continuous treatment (24 h or 48 h) test. However, in the short-period test with rat liver S9 mix, LFO induced structural chromosomal aberrations at concentrations higher than 0.6 mg/mL. A bone marrow micronucleus test using male F344 rats was initially conducted. The animals were dosed by oral gavage at doses up to 5000 mg/kg/day. No significant or dose-dependent increases in the frequency of micronucleated polychromatic erythrocytes (MNPCE) were observed and the high dose suppressed the ratio of polychromatic erythrocytes (PCE) to total erythrocytes. Subsequently, a liver and peripheral blood micronucleus test using male F344 rats was conducted. No micronuclei induction either in hepatocytes or PCE was observed even at the highest dose of 5000 mg/kg/day. From the findings obtained from the genotoxicity assays performed in this study and the published pharmacokinetic studies of LFO, it appears unlikely that dietary consumption of LFO will present any genotoxic hazard to humans.     

Micronucleus induction by diuron in mouse bone marrow

Diuron, a widely used substituted urea herbicide, induced the formation of micronuclei in bone marrow cells of Swiss mice. A single i.p. dose of 340 mg/kg b.w. diuron which is maximum tolerated dose (MTD) increased significantly the number of micronuclei at 30 h and 48 h time period. The dose of 170 mg/kg b.w. also induced the micronuclei formation in the above time period. However, a dose of 85 mg/kg b.w. was ineffective at the time periods studied. No induction of micronuclei was observed at 72 h time period after all the doses of diuron studied as compared to the solvent control. The diuron-induced frequency of micronucleated erythrocytes was independent of the sex of the test animals.     

Genotoxic effects of (-)-cubebin in somatic cells of mice

(?)‐Cubebin belongs to the dibenzylbutyrolactone lignan group, which is widely distributed in the plant kingdom. Because this compound shows interesting biological activities, it is extremely important to evaluate its possible genotoxic activity to allow its safe use in humans. Thus, the present study was performed to investigate the genotoxicity potential activity of (?)‐cubebin assessed by two assays: micronucleus in bone marrow cells and comet test in peripheral blood leukocytes of Swiss mice. In the (?)‐cubebin dose range‐finding assays, the maximum tolerated dose was greater than 2000 mg kg^?1. The compound was administered by an oral route at single doses of 250, 500 and 2000 mg kg^?1 body weight. Cytotoxicity was assessed by scoring 200 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). Under our experimental conditions, micronucleus and comet assays, respectively, showed that (?)‐cubebin caused dose‐related clastogenic and genotoxic effects in the somatic cells investigated. PCE/NCE ratio showed no cytotoxicity for the three doses of the compound. The data suggest caution in the ingestion of (?)‐cubebin by humans, especially at high doses. Copyright © 2010 John Wiley & Sons, Ltd.     

Genotoxicity assessment of the antimalarial compound artesunate in somatic cells of mice

Artesunate is a derivate of artemisinin that is both an antimalarial agent and acts cytotoxically on tumor cells. Despite its therapeutic use, its in vivo genotoxic potential has still not been evaluated. This study, therefore, was an investigation into the effects of a single oral administration of artesunate with an in vivo comet assay that analyzed leukocytes from peripheral blood and liver cells, and a micronucleus (MN) assay of bone marrow cells from male Swiss mice. The artesunate was administered by oral gavage at doses of 5, 50 and 100 mg/kg. Cytotoxicity was assessed by scoring 200 consecutive polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE/NCE ratio). The results demonstrate that artesunate induced significant DNA damage only in liver cells and that high doses of artesunate caused an increase in the mean number of micronucleated polychromatic erythrocytes (MNPCE). Under our experimental conditions, artesunate showed weak genotoxic effects at low doses and clastogenic effects at high doses. The PCE/NCE ratio indicated no cytotoxicity. The data obtained suggest caution about either continuous or high-dose use of artesunate by humans.     

The toxicity of behenyl alcohol. I. Genotoxicity and subchronic toxicity in rats and dogs

The genotoxic potential of behenyl alcohol, a saturated long-chain (C22:0) fatty alcohol, was examined in the Ames Salmonella typhimurium reverse mutation assay, the gene mutation, and chromosome aberrations assays in Chinese hamster V79 cells, and the micronucleus assay in NMRI mice. Behenyl alcohol did not increase the number of revertants per plate compared to controls in the S. typhimurium assay, with or without metabolic activation. No significant increases in the number of mutant colonies or in structural chromosome aberrations were observed in Chinese hamster V79 cells. In addition, behenyl alcohol did not increase the frequency of bone marrow polychromatic erythrocyte (PCE) micronuclei in mice in vivo. In two subchronic toxicity studies, CD rats and beagle dogs were administered behenyl alcohol by oral gavage for at least 26 weeks at doses of 0, 10, 100, or 1000 mg behenyl alcohol/kg body weight/day for rats and 0, 20, 200, or 2000 mg behenyl alcohol/kg body weight/day for dogs. Adverse effects were not observed following gross and histopathological evaluations of dosed rats. Compound-related effects in dogs were limited to observations of pale feces, indicative of unabsorbed behenyl alcohol, at doses of 2000 mg/kg body weight/day. There were no histopathological changes observed in dogs dosed with behenyl alcohol. The no-observed-adverse-effect-level (NOAEL) for behenyl alcohol was 1000 mg/kg body weight/day for rats, and 2000 mg/kg body weight/day for dogs, the highest doses used in these studies.     

The relationship between stress protein induction and the oxidative defense system in the rat hippocampus following kainic acid administration

The time and dose-dependent effects of kainic acid (KA) induced excitotoxicity on the oxidative defense system and the relationship to the induction of stress proteins were investigated in the rat hippocampus. Male Long-Evans rats were injected subcutaneously with 5.0, 7.5, or 10 mg KA/kg. Rats were sacrificed and the hippocampus removed and processed for biochemical and electrophoretic analysis. The activity of glutathione peroxidase (GPx) increased significantly at the 5 mg KA/kg dose, while malondialdehyde (MDA) levels significantly increased at 7.5 mg KA/kg when measured at 24 h. A dose of 10 mg KA/kg depleted significantly hippocampal glutathione (GSH) levels at 8, 16 and 24 h post-treatment while GPx activity was increased significantly at 2, 4, 8 and 16 hr post-treatment. The 10 mg KA/kg increased significantly hippocampal MDA levels at 2 h post-treatment and decreased significantly thereafter. The induction of stress proteins increased in a dose and time dependent manner. The expression of Hp72 and Hsp32 increased significantly at 16 h with a maximum induction observed at 24 h post-treatment. The data suggests that KA toxicity is mediated through the formation of reactive oxygen species resulting in alterations in the oxidative defense system. The expression of stress proteins following KA administration may reflect a concomitant but alternate response to excitotoxic events.     

In vivo and in vitro studies on the genotoxicity of cadmium chloride in mice

The genotoxic effect of cadmium chloride was evaluated in chromosomes of experimental mice using in vivo and in vitro studies. In vivo the induction of micronuclei, sister chromatid exchange in mouse bone marrow and chromosomal aberrations in both somatic and germ cells was investigated. Doses 1.9, 5.7 and 7.6 mg kg(-1) body wt. (single i.p. treatment) induced a significant and dose-dependent increase in the percentage of polychromatic erythrocytes with micronuclei. Such a percentage reached 2.1% with the highest tested dose, compared with 0.57% for the control (non-treated) and 2.2% for mitomycin c as the positive control. The dose of 1.9 mg kg(-1) body wt. had no significant effect with respect to sister chromatid exchange (SCE) but the doses of 5.7 and 7.6 mg kg(-1)body wt. increased the frequency of SCEs significantly. The frequency of SCE reached 7.35 +/- 0.26 per cell after treatment with the highest tested dose, which is a less than twofold increase compared with the control frequency of 4.6 +/- 0.42 per cell. However mitomycin c induced a much higher effect (12.1 +/- 0.73). Cadmium chloride also induced a significant increase in the percentage of chromosomal aberrations in mouse bone marrow at the doses of 5.7 and 9.5 mg kg(-1) body wt. (single i.p. treatment). The effect is a function of cadmium chloride concentration. Moreover, cadmium chloride induced its maximum effect concerning the induction of chromosomal aberrations in mouse bone marrow cells 24 h after treatment, compared with 12 and 48 h. In germ cells, chromosomal aberrations were observed in mouse spermatocytes 12 days post-treatment with the dose of 5.7 mg kg(-1) body wt. Moreover, a pronounced reduction in the number of spermatocytes was observed after administration of cadmium chloride (0.9, 1.9 and 5.7 mg kg(-1) body wt.) In in vitro studies, the three tested concentrations of 10, 15 and 20 microgram ml(-1) cadmium chloride induced a statistically significant increase in the frequency of SCEs in cultured mouse spleen cells. The concentrations of 15 and 20 microgram ml(-1) also induced chromosomal aberrations in mouse spleen culture. The ability of vitamin C (l-ascorbic acid) to minimize the incidence of chromosomal aberrations induced by cadmium chloride in cultured mouse spleen cells was investigated. Vitamin C at the concentrations of 3 and 6 microgram ml(-1) significantly minimized the percentage of aberrant cells induced by cadmium chloride.     

Nandrolone androgenic hormone presents genotoxic effects in different cells of mice

Nandrolone is an androgenic–anabolic steroid (AAS) with diverse medical applications but taken indiscriminately by some to rapidly increase muscle mass. The aim of this study was to evaluate the genotoxic and clastogenic potential of nandrolone (deca‐durabolin®) in vivo in different cells of mice, using the comet assay and micronucleus test, respectively. The animals received subcutaneous injection of the three doses of the steroid (1.0, 2.5 and 5.0 mg kg^?1 body weight). Cytotoxicity was assessed by scoring 200 consecutive total polychromatic (PCE) and normochromatic (NCE) erythrocytes (PCE–NCE ratio). The results showed a significant dose‐related increase in the frequency of DNA damage in leukocytes, liver, bone marrow, brain and testicle cells at the three tested doses and a significant increase of the micronucleated polychromatic erythrocytes at all tested doses. Under our experimental conditions, the nandrolone steroid hormone showed genotoxic and clastogenic effects when administered subcutaneously to mice. Copyright © 2011 John Wiley & Sons, Ltd.