Genotoxicity, DNA damage and sperm defects induced by vinblastine.

BACKGROUND: The treatment with chemotherapy may develop secondary tumors as a result of chemo genotoxicity. Sperm defects is another complication associated with chemo treatment. In this study the genotoxicity of vinblastine (VB) was estimated in both somatic and germ cells. MATERIALS: 85 mice were taken. Four single doses of VB at 3, 4.5, 6 and 10 mg/kg and three successive doses at 3, 4.5 and 6 mg/kg were taken for estimation of chromosomal aberrations (CAs). Four single doses of VB were involved in estimating the DNA fragmentation, and comet assay. For sperm abnormalities mice were injected with three successive doses of VB at 3, 4.5, and 6 mg/kg. RESULTS: The results demonstrated a significant frequency of DNA fragmentation in spleen cells and in the percentage of CAs in bone marrow. Numerical and structural aberrations were recorded with a pronounced number of polyploidy metaphases which reached (11.60%) after treatment with 6 mg/kg for three successive days vs zero for control. VB also induced a significant percentage of CAs in spermatocytes in the form of univalent. Sperm defects in the form of coiled tail, absence of acrosome and shapeless head and a significant DNA damage in the testes were recorded. The frequency of sperm abnormalities reached 11.06 ± 0.14 after treatment with highest tested dose (6 mg/kg) vs 3.04 ± 0.19 for control. CONCLUSION: VB is genotoxic in somatic and germ cells. Sperm defects induced by VB are of serious concern to future generations and may affect the fertility of cancer survivors.

Protective role of Codiaeum variegatum against genotoxicity induced by carmustine in somatic and germ cells of male mice.

BACKGROUND: Carmustine (Cr) is an important chemotherapeutic drug, widely used in the treatment of brain tumors. Herein, the protective role of Codiaeum variegatum leaves ethyl acetate fraction was determined against genotoxicity of Cr. The technique HPLC-qTOF-MS/MS was used to identify the constituents in C. variegatum. MATERIALS: 90 male mice were used to evaluate micronuclei (MPCEs) in bone marrow, chromosomal aberration (CAs) in bone marrow and mouse spermatocytes, sperm abnormalities, and gene expression (qRT-PCR). The following groups were included, I: Negative control (ethanol 30%), II: Positive control (i.p injected once with 30 mg/kg Cr), III: Control orally treated with C. variegatum at 500 mg/kg, four days. IV-VI: treated with 100, 300, and 500 mg/kg of the plant (4 days) plus a single dose of Cr. RESULTS: In bone marrow, Cr induced significant increase in MPCEs and CAs by 3 and 7-folds respectively over the control. Cr also induced a significant percentage of CAs in spermatocytes in meiosis in the form of univalent (X-Y and autosomal univalent) and also a significant percentage of morphological sperm abnormalities was recorded. A large number of coiled tail abnormalities were detected indicating the effect of Cr in sperm motility. Cr induced an overexpression of p53 gene. C. variegatum mitigated all deleterious genotoxic effects of Cr. Chemical analysis showed that flavones (35.21%) and phenolic acids (17.62%) constitute the main components. CONCLUSIONS: The results indicated that Cr is genotoxic in both somatic and germ cells. The active components in C. variegatum together participate in the obtained protective role.

Evaluation of the cytotoxic, anticancer, and genotoxic activities of Acacia nilotica flowers and their effects on N-methyl-N-nitrosourea-induced genotoxicity in mice.

PURPOSE: In this study, two main research objectives were examined: (1) the cytotoxic and anticancer activities of the aqueous methanol extract from Acacia nilotica flowers on three human cancer cells, namely lung A549, breast MCF-7, and leukemia THP-1 cells, and (2) the genotoxic effects of A. nilotica extract and its influence on DNA damage induced by N-methyl-N-nitrosourea (MNU) in mice. METHODS: Mice were orally treated with A. nilotica extract (200, 500, and 800 mg/kg for 4 days) with or without MNU (80 mg/kg intraperitoneally for 24 h). RESULTS: In vitro experiments showed that A549 cells were the most sensitive to A. nilotica extract among the tested cell lines. A. nilotica extract inhibited A549 cell proliferation by blocking the cell cycle at the G2/M phase and accumulating apoptotic cells in the sub-G0/G1 phase in A549 cells. In vivo experiments showed that MNU induced positive and negative genotoxicity in bone marrow cells and spermatocytes, respectively. Negative genotoxicity was observed in A. nilotica extract-treated groups only. However, A. nilotica extract (800 mg/kg) remarkably increased comet tail formation in bone marrow cells. Unexpectedly, the absence of antigenotoxicity was observed in three cotreated groups with A. nilotica extract and MNU compared with the MNU-treated group. Astonishingly, cotreatment with MNU and A. nilotica extract at a dose above 200 mg/kg remarkably increased micronucleus and comet tail formation in bone marrow cells compared with the MNU-treated group. CONCLUSIONS: A. nilotica extract possessed anticancer activity with relative genotoxic effects at high doses.

Evaluation of the Anti-Cancer/Anti-Mutagenic Efficiency of Lavandula officinalis Essential Oil.

OBJECTIVE: Lavender oil is of a great economic importance. It has many biological and pharmacological activities. The present study aimed to identify the chemical constituents of the essential oil of Lavandula officinalis (LAEO) by using GC/MS analysis. Its genotoxicity, anti-genotoxicity and histopathological activities against the chemotherapeutic drug cyclophosphamide (CP) was investigated. The study also evaluated its anticancer activities against six human cancer cell lines: hepatocellular carcinoma (HepG2), Prostate (PC3), Lung carcinoma (A549), Skin cancer (A431), Colon cancer (HCT116) and Breast cancer (MCF7). METHODS: The genotoxicity was determined using: micronucleus, chromosomal aberration, and comet assays. The histopathological study included liver. The examined groups were control negative, control plant, control positive (CP), and 3 combined groups received LAEO at different concentrations plus CP. RESULTS: GC/MS analysis recorded 16 components. The principals were: linalool and linalyl acetate. The results indicated the safety of LAEO. It also attenuates genotoxicity and deleterious histopathological effects of CP in a dose-dependent manner. LAEO has a highly cytotoxic effect on HepG2 and A549 cell lines with 100% death at 100µg/ ml with IC50 67.8 and 12 µg/ ml, respectively. Its activity on other cell lines was weak. CONCLUSION: The essential oil of Lavandula officinalis has anticancer and anti-mutagenic effect.

Inhibitory activity of flaxseed oil against CdCl2 induced liver and kidney damage: Histopathology, genotoxicity, and gene expression study.

The present work evaluated the effect of flaxseed oil (FO) against toxicity induced by cadmium chloride (CdCl2) in the mouse liver and kidney. Male Swiss albino mice were treated with CdCl2 (4.5 mg/kg, intraperitoneally) with or without FO at three concentrations (4, 8, 12 mL/kg, orally) for two consecutive weeks. To analyze the effects of FO, we used the following techniques: (1) histopathological examination; (2) comet assay; (3) RT-PCR gene expression analysis of tumor necrosis factor (TNF-α) and tumor suppressor protein (p53); and (4) immunohistochemical analysis of caspase-9 protein expression. The gas chromatography-mass spectrometry results showed that FO had a high content of unsaturated fatty acids including, oleic acid, linolenic acid, and linoleic acid. Oral supplementation with FO (12 mL/kg) resulted in a normal histological appearance without alteration in the DNA integrity and gene expression of TNF-α, p53, and caspase-9 in liver and kidney tissues. As expected, CdCl2 remarkably induced loss of histological integrity, increased DNA comet formation, increased TNF-α and p53 mRNA expression levels and increased the immunoreactivity of caspase-9 expression. When FO was given before administration of CdCl2, these histopathological defects were reversed; necrosis, degeneration, inflammatory cell infiltration, hemorrhage, Kupffer cells, and pyknotic cells were all reduced. These histological improvements induced by FO were accompanied by reduced DNA breakage, downregulated mRNA expression of TNF-α and p53, and downregulated immunohistochemical expression of caspase-9 protein. In conclusion, FO and its constituents may act as signaling molecules and modify the expression of genes involved in proinflammatory cytokine production (TNF-α), cell cycle arrest (p53), and apoptosis (caspase-9), thereby improving biological activities and health.

Genotoxicity and sperm defects induced by 5-FU in male mice and the possible protective role of Pentas lanceolata-iridoids.

5-Fluorouracil (5-FU) is a widely used antineoplastic drug. In this work, a comprehensive study was performed to detect the extent of chromosomal damage and morphological sperm defects induced by 5-FU in male mice and the possible protective role of the iridoids-rich fraction of Pentas lanceolata leaves (IFPL). Six main groups were examined in micronucleus and chromosomal assays: I- control negative, II- control positive (i.p. treated with single dose of 75 mg/kg 5-FU), III- control plant (orally administrated IFPL, 300 mg/kg, 5 consecutive days), and IV-VI- treated with IFPL (100, 200 and 300 mg/kg, 5 consecutive days) plus 5-FU (i.p. treated at the last day). Samples were taken 24 h post treatment. The study of morphological sperm anomalies, single and repeated treatments were examined and samples were taken after 35 days from the 1st treatment. In bone marrow, 5-FU induced a significant increase in the micro-nucleated polychromatic erythrocytes, chromosome anomalies (CAs) and also cytotoxic effects. A significant percentage of CAs was recorded in spermatocytes after 5-FU treatment reached 22.80 ± 1.32 vs 4.20 ± 0.37 for control (mainly X-Y univalent, 90%). IFPL was recorded to be non-mutagenic in all tests examined. In addition, it alleviated the previous defects in a dose-dependent manner. A significant and dramatic increase in the percentage of morphological sperm defects was recorded after single and repeated treatments with 5-FU reached 13.24 ± 0.24, 30.42 ± 0.32 respectively vs 2.56 ± 0.14 for control. Amorphous head-sperm and sperm with coiled tail were the most pronounced types of abnormalities. Significant protection was detected with the highest tested dose of IFPL. In conclusion: 5-FU demonstrated to be a genotoxic agent. Its genotoxicity in germ cells is serious and may lead to reproductive toxicity, infertility or heritable defects. The results also demonstrated the biosafety of IFPL and its possible protective role in combined treatment with 5-FU.

Inhibitory activity of black mulberry (Morus nigra) extract against testicular, liver and kidney toxicity induced by paracetamol in mice.

Black mulberry (Morus nigra) leaves is broadly used in traditional medicine worldwide. However, there are no scientific reports regarding testicular protection, hepato-and nephroprotective activities of M. nigra leaves. The present investigation was assessed the protective mechanism by which methanol extract from M. nigra leaves suppressed the damaging effects induced by paracetamol (APAP) in different mouse tissues. Male mice were orally given APAP (500 mg/kg) with or without M. nigra extract (150, 300, and 500 mg/kg) for four consecutive days. The results showed that crude extract possessed potent antioxidant activity (EC50 = 42.97 µg extract/mL) due to the presence of a high amount of polyphenol and flavonoid compounds. Gallic acid, chlorogenic acid, catechin, and rutin were isolated from the n-butanol fraction of M. nigra extract. Unexpectedly, oral administration of APAP did not induce chromosomal aberrations in mouse bone marrow; however, it produced damaging effects on testis, liver, and kidney tissues. Interestingly, M. nigra extract suppressed APAP-induced genotoxicity by lowering meiotic chromosomal aberrations in spermatocytes, morphological sperm abnormalities, and % DNA damage in comet tail in the liver and kidney tissues. The altered levels of glutathione S transferase activity, lipid peroxidation, liver, and kidney functions were significantly reversed when M. nigra was given to APAP group. The restoring of the histo-architectural distortions and decreasing over-expression of p53 protein as determined by immunohistochemistry in the liver, kidney, and testis sections were strengthened the protective activity of M. nigra extract. Conclusion, the bioactive components in the leaves of black mulberry appear to be a good candidate for genetic protection, treatment of oxidative stress-induced organotoxicity.

Carbon tetrachloride induced hepato/renal toxicity in experimental mice: antioxidant potential of Egyptian Salvia officinalis L essential oil.

The present research designed to assess the protective role of Salvia officinalis essential oil (SO) against carbon tetrachloride (CCl4)-induced liver and kidney damage in mice. This is evidenced by estimation of antiradical scavenging activity of SO using DPPH assay, biochemical markers, histological investigation of liver and kidney sections, and comet assay. Mice were given CCl4 (1.2 mL/kg for 24 h or 0.8 mL/kg for 2 weeks, 3 times/week) and with or without SO (0.1, 0.2, and 0.4 mL/kg, for 2 week, 5 times/week). The findings demonstrated that both acute and subacute treatment with CCl4 alone had adverse side effects on liver and kidney of mice. These effects were evidenced by a significant increase in serum hepatic enzymes (ALT, AST, ALP, LDH, and G-GT), bilirubin, and renal function markers (blood urea, creatinine). Toxic effect of CCl4 was accompanied by a decline in the serum total protein, albumin, globulin, and prothrombin (%). CCl4 induced oxidative stress as evidenced by increasing serum lipid peroxidation (LPO) along with decreasing serum total glutathione S transferase (GST). A remarkable increase in hepatic DNA strand breakages and histopathological distortion in liver and kidney specimens were observed in CCl4-intoxicated groups. Ultrastructurally, hepatocytes exhibited irregular nuclei, vacuolated cytoplasm, and distorted microorganelles. Essential oil form S. officinalis possessed antiradical scavenging (EC50 = 4602 µg/mL) lower than ascorbic acid (EC50 = 5.9 µg/mL). This oil was effectively exhibited hepato-nephroprotective activity especially at its higher concentrations in co-treated groups (SO plus CCl4). The activity of SO was associated with lowering the liver enzymes, bilirubin, urea, and creatinine, along with increasing total protein, albumin, globulin, and prothrombin. The increase in GST content and the decrease in LPO and DNA breakage levels, alongside repairing the histo-architectural distortions further confirmed the protective activity of SO. SO is a potential candidate for counteracting hepato/renal injury associating CCl4. This effect may occur via antioxidant defense mechanism which in part related to the complexity of its chemical constituents.

Genotoxicity of carbon tetrachloride and the protective role of essential oil of Salvia officinalis L. in mice using chromosomal aberration, micronuclei formation, and comet assay.

The present work was conducted to evaluate the genotoxic effect of carbon tetrachloride (CCl4) in mouse bone marrow and male germ cells. The safety and the modulating activity of sage (Salvia officinalis L.) essential oil (SEO) against the possible genotoxic effect of CCl4 were also evaluated. A combination of in vivo mutagenic endpoints was included: micronucleus (MN), apoptosis using dual acridine orange/ethidium bromide (AO/EB) staining, comet assay, chromosomal aberrations (CAs), and sperm abnormalities. Histological examination of testis tissues was also studied. The extracted SEO was subjected to gas chromatography-mass spectrometry (GC-MS) for identifying its chemical constituents. Safety/genotoxicity of SEO was determined after two consecutive weeks (5 days/week) from oral treatment with different concentrations (0.1, 0.2, and 0.4 mL/kg). For assessing genotoxicity of CCl4, both acute (once) and subacute i.p. treatment for 2 weeks (3 days/week) with the concentrations 1.2 mL/kg (for acute) and 0.8 mL/kg (for subacute) were performed. For evaluating the protective role of SEO, simultaneous treatment with SEO plus CCl4 was examined. In sperm abnormalities, mice were treated with the subject materials for five successive days and the samples were collected after 35 days from the beginning of treatment. Based on GC-MS findings, 22 components were identified in the chromatogram of SEO. The results demonstrated that the three concentrations of SEO were safe and non-genotoxic in all the tested endpoints. Negative results were also observed in bone marrow after acute and subacute treatment with CCl4. In contrast, CCl4 induced testicular DNA damage as evidenced by a significant increase of CAs in primary spermatocytes, sperm abnormalities, and histological distortion of testis. A remarkable reduction in these cells was observed in groups treated with SEO plus CCl4 especially with the two higher concentrations of SEO. In conclusion, SEO is safe and non-genotoxic under the tested conditions and can modulate genetic damage and histological alteration induced by CCl4 in the testes.

The modifying effect of selenium and vitamins A, C, and E on the genotoxicity induced by sunset yellow in male mice.

The use of food additives in various products is growing up. It has attracted the attention towards the possible correlation between the mutagenic potential of food additives and various human diseases. This work evaluated the protective role of selenium and vitamins A, C and E (selenium ACE)(1) against the genotoxic effects induced by a synthetic food additive, sunset yellow, in mice. Six groups were studied including two control groups (negative and positive control), two groups are given single dose of sunset yellow (either 0.325, 0.65 or 1.3mg/kg body weight(2) alone or with selenium ACE) and two groups are given sunset yellow daily for 1, 2 or 3 weeks (0.325mg/kg b.wt./day alone or with selenium ACE), respectively. The study examined the induction of sister chromatid exchanges (SCE's)(3) in bone-marrow cells, chromosomal aberration in somatic (bone-marrow) and germ cells (spermatocytes) after single and repeated oral treatment, and the induction of morphological sperm abnormalities. The results showed that sunset yellow had genotoxic effects as indicated by increased frequency of SCE's, by chromosomal aberrations in both somatic and germ cells, and by increased morphological sperm abnormalities and DNA fragmentation. The results also indicated that the oral administration of selenium ACE significantly reduced the genotoxic effects of sunset yellow, a result that may support the use of antioxidants as chemopreventive agents in many applications.

Studies on the genotoxic effect of beryllium chloride and the possible protective role of selenium/vitamins A, C and E.

The genotoxic potential of beryllium chloride (BeCl2) was evaluated in vivo in mice using different endpoints. Chromosomal aberrations in bone marrow cells and in spermatocytes as well as sperm abnormalities were determined in the tested mice. The protective role of an orally administered drug consisting of selenium and vitamins A, C and E (selenium-ACE) was also studied. For analysis of chromosomal aberrations, both single and repeated oral treatments for a period of 3 weeks were performed. The doses used were 93.75, 187.50, 375, and 750 mg BeCl2/kg bw, which corresponds to 1/16, 1/8, 1/4, and 1/2 of the experimental LD50. BeCl2 induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells, with a dose- and time-response. The percentage of induced chromosomal aberrations was significantly reduced in all BeCl2-treated groups after oral administration of selenium-ACE. Beryllium chloride also induced a significant increase in the percentage of abnormal sperm. This percentage reached values of 9.62 +/- 0.32 and 5.56 +/- 0.31 in mice treated with the highest test dose of BeCl2 and with BeCl2+selenium-ACE, respectively, compared with 1.96 +/- 0.14 for the control. In conclusion, the results demonstrate the genotoxic effect of beryllium chloride and confirm the protective role of selenium-ACE against the genotoxicity of beryllium chloride.

The protective role of thiola and soybean seeds against the genotoxicity induced by potassium dichromate in mice.

The genotoxic potential of potassium dichromate (K(2)Cr(2)O(7)) was evaluated in vivo in mice using different mutagenic end points. Chromosomal aberrations in bone-marrow and spermatocytes as well as sperm abnormalities in the tested mice were determined. The doses used were 3, 6, 12 mg K(2)Cr(2)O(7)kg(-1) body weight which correspond to 1/16, 1/8, 1/4 the experimental LD(50), respectively. The protective roles of i.p. injection with thiola (a synthetic sulfhydryl compound) at 20 mg kg(-1) body weight and feeding treatment with soybean seeds (30% of the diet) were also studied. For chromosomal aberration analysis, subacute treatment for a period of 3 weeks were performed. All the tested doses of K(2)Cr(2)O(7) induced a statistically significant increase in the percentage of chromosomal aberrations in both somatic and germ cells with dose and time relationships. The percentage of the induced chromosomal aberrations was significantly minimized in all groups of mice i.p. treated with thiola or fed soybean seeds during the period of treatment. Potassium dichromate also induced a significant increase (P<0.01) in the percentage of abnormal sperms at the doses 6 and 12 mg kg(-1) body weight. Such percentage reached 7.52+/-0.45, 5.50+/-0.53 and 4.28+/-0.45 in mice treated with the highest tested dose of K(2)Cr(2)O(7), K(2)Cr(2)O(7) and thiola; K(2)Cr(2)O(7) and soybean, respectively compared with 2.14+/-0.33 for the control. In conclusion, the results demonstrate the genotoxic effect of potassium dichromate in mice. The results also confirm the protective role of thiola and soybean seeds against the genotoxicity of potassium dichromate.

Cytogenetic studies on the effect of feeding mice with stored wheat grains treated with malathion.

The cytogenetic effect of malathion residues in wheat grains stored for different periods of time (4, 12, 24 weeks) was evaluated in Swiss mice. The studies included: (1) chromosomal aberrations analysis in bone-marrow and spermatocyte cells; (2) chromosomal aberrations and sister chromatid exchange (SCE) analysis in spleen cell culture from mice fed with stored wheat grains. The tested doses were 8.36 (applied dose), 25.08 and 41.80 mg malathion kg(-1) wheat grains. The results demonstrated that the cytogenetic effect induced in different mouse tissues by malathion residues was dose-dependent and increased with increasing of both feeding and storage periods. Feeding mice with wheat grains stored for 4 weeks had a non-significant effect with respect to the induction of chromosomal aberrations or SCEs. Significant chromosome damage and increase of SCEs were observed in mice fed with wheat grains stored for 12 weeks. The maximum effect was recorded in mice fed for 12 weeks with the grains treated with the highest tested dose and stored for 24 weeks. However, mitomycin C i.p.-injected in mice at 1 mg kg(-1) body weight (b.w.) (positive control) induced a higher effect. The percentage of chromosome aberrations reached 13.60+/-0.98, 13.60+/-0.77 and 11.73+/-0.98 (P<0.01) in bone-marrow, cultured spleen cells and spermatocytes, respectively. The significant increase of abnormalities in spermatocytes was seen for univalent formation only, predominantly of the sex chromosomes. The frequency of SCEs was 10.76+/-0.62 per cell (P<0.01) in cultured spleen cells compared with 5.46+/-0.45 per cell for control and 14.66+/-0.54 per cell for the positive control. The obtained results indicate that malathion residues in stored wheat grains have potential genotoxic effect in mice under the conditions tested.