CP-induced DNA Damage Research Articles

Melatonin Ameliorates Cyclophosphamide-Induced Spermatogenesis Disorder by Reducing Pyroptosis

As a chemotherapeutic drug, cyclophosphamide (CP) has a negative impact on male fertility due to its reproductive toxicity. Melatonin (Mel) promotes the male reproductive system and increases testosterone synthesis. This study is aimed at exploring the molecular mechanism of Mel as a protector of male fertility against CP-induced cytotoxicity. A CP toxicity model was established in adult ICR male mice by intraperitoneal injection of 100 mg/kg CP every other day for a week. Protective effects of Mel on the testis from CP-induced damage were evaluated using four groups of ICR male mice that received intraperitoneal injections of normal saline, 100 mg/kg CP, 10 mg/kg Mel, or the same dosage of CP and Mel, respectively. Testis morphology was observed by hematoxylin and eosin (HE) staining. Sperm quality parameters were evaluated, and sperm function was studied by in vitro fertilization (IVF). Proliferation, meiosis, and pyroptosis markers were examined by western blot. Results showed that CP treatment induced testis toxicity in a time-dependent manner with the most severe damage to the testis at two weeks post CP treatment. CP-treated mice showed reduced testicular weight and impaired spermatogenesis by downregulating PCNA and SYCP3, reduced serum testosterone levels, decreased sperm counts and motility, increased seminiferous tubule vacuolization, and oxidative damage to spermatogenic cells. All these effects, apart from testicular weight, could be ameliorated by Mel administration. The IVF experiment revealed that CP treatment reduced the rates of sperm fertilization and blastocyst development, which were also enhanced by Mel. Mel-treated mice also showed increased expression of proliferation-associated protein PCNA and meiosis-associated proteins REC8, STRA8, and SYCP3, which were all reduced by CP. Furthermore, Mel inhibited the pyroptosis of spermatogenic cells by reducing GSDMD and IL18 expression. In conclusion, this study indicated that Mel might protect the testis from CP-induced DNA damage to germ cells through the alleviation of pyroptosis.

Evaluation of Genotoxic Effect and Antigenotoxic Potential against DNA Damage of the Aqueous and Ethanolic Leaf Extracts of Annona muricata Using an In Vivo Erythrocyte Rodent Micronucleus Assay.

Annona muricata have been extensively used in traditional medicine to treat multiple diseases, including cancers. This study evaluated the genotoxic potential and antigenotoxic activities of A. muricata aqueous and ethanolic leaf extracts by employing an in vivo erythrocyte rodent micronucleus assay. Different doses (187.5, 375, and 750 mg/kg) of both extracts were administered orally for 5 days alone and combined with cyclophosphamide (CP, 60 mg/kg) to BALB/c mice. Also, it was administered orally to Wistar rats for 5 days through the final stage of gestation. No genotoxic or cytotoxic effects were observed in the two adult rodent models when A. muricata was administered orally nor in newborn rats transplacentally exposed to the extracts. Moreover, A. muricata aqueous and ethanolic leaf extracts demonstrated a protective effect against CP-induced DNA damage. Due to its lack of genotoxic effect and its capacity to decrease DNA damage, A. muricata is likely to open an interest field regarding its potential safe use in clinical applications.

Ethanolic extract of Moringa oleifera leaves alleviate cyclophosphamide-induced testicular toxicity by improving endocrine function and modulating cell specific gene expression in mouse testis

Ethnopharmacological relevanceMoringa oleifera Lam. is known for its nutritional and ethno medicinal values due to the presence of wide array of phytochemicals with multiple biological activities. We have previously reported that ethanolic extract of Moringa oleifera leaves (MOE) ameliorated cyclophosphamide (CP)-induced testicular toxicity and improved functional integrity of spermatozoa as well as spermatogenic cells. Aim of the studyThe present study was planned to investigate whether the mitigation of CP-induced testicular toxicity by MOE is mediated via modulation of endocrine profile, genes associated with function of different cell types and enhancement of DNA repair response in spermatogonial cells. Materials and methodsAdult Swiss albino mice (8 week) were injected with CP (100 mg/kg, one dose in a week for 3 weeks) and MOE (100 mg/kg, 5 doses in a week for 4 weeks) either alone or in combination intraperitoneally. At 35 day post CP injection (first dose), the functional characteristics such as count, motility, head morphology and DNA integrity were assessed in epididymal spermatozoa. Key reproductive hormones like testosterone, follicle stimulating hormone (FSH) and Inhibin B concentration were analyzed in serum and testis. In addition, mRNA expression of genes pertaining to the function of Leydig, Sertoli and spermatogonial cells as well as antioxidant enzymes were evaluated in the testis. To understand the DNA damage and repair process in germ cells, prepubertal (2 week) mice were administered with single dose of CP (200 mg/kg) and/or MOE (100 mg/kg) and analyzed for expression of DNA damage (γ-H2AX, P53 and Caspase3) and repair genes (Rad51 and Ku80) in isolated spermatogonial cells at various time points after treatment. ResultsCP administration resulted in decrease in count, motility and increase in morphological defects and DNA damage in spermatozoa. Testosterone level was marginally decreased while there was a significant increase in FSH (p < 0.001) and decrease in inhibin B (p < 0.05) observed in CP treated mice. Administration of MOE prior to CP, improved sperm functional characteristics, decreased FSH and increased inhibin B levels. Expression of Abp was down-regulated while Transferrin, Fshr and Gata4 (Sertoli cell specific genes) were up-regulated in testis treated with CP. Administration of CP down-regulated the expression of Oct4 and Ddx4 (Spermatogonia specific genes). MOE administration was shown to ameliorate CP-induced damage by modulating the expression of genes specific to Sertoli and spermatogenic cells. Furthermore, MOE treatment reduced CP-induced DNA damage as evident from lower percentage of γ-H2AX positive spermatogonial cells. ConclusionAdministration of MOE mitigated CP-induced testicular damage by improving blood and, intra-testicular hormonal milieu as well as modulating the expression of genes pertaining to Sertoli and spermatogonial cells.

A Supersaturated Oxygen Emulsion for the Topical Treatment of Ocular Trauma.

Roughly 13% of all battlefield injuries include some form of ocular trauma. Ocular tissue preservation is critical for wound healing for warfighters with ocular injuries. Our team hypothesized that oxygen plays a vital role in ocular tissue preservation and wound healing and has developed a supersaturated oxygen emulsion (SOE) for the topical treatment of ocular trauma. The partial pressure of oxygen (PO2) was measured in the SOE. Safety and efficacy studies were carried out in primary human corneal epithelial (HCE) cells, as the outermost layer is the first barrier to chemical and mechanical injury. Western blot, scratch assay, and MTT assays were conducted to determine the effect of the SOE on various molecular markers, the rate of scratch closure, and cellular viability, respectively. Data indicate that the SOE releases oxygen in a time-dependent manner, reaching a partial pressure within the emulsion over four times atmospheric levels. Studies in HCE cells indicate that application of the SOE does not lead to DNA damage, promote cell death, or hinder the rate of scratch closure and enhances cellular viability. Preliminary studies were carried out with chloropicrin (CP; developed as a chemical warfare agent and now a commonly used pesticide) as a chemical agent to induce ocular injury in HCE cells. CP exposures showed that SOE treatment reverses CP-induced DNA damage, apoptotic cell death, and oxidative stress markers. Maintaining adequate tissue oxygenation is critical for tissue preservation and wound repair, especially in avascular tissues like the cornea. Further studies examining the application of the SOE in corneal injury models are warranted.

Modulation of hepatic ABC transporters by Eruca vesicaria intake: Potential diet-drug interactions

The aim of this work was to evaluate whether oral administration of Eruca vesicaria, a species of rocket cultivated in Argentina, could modify cyclophosphamide (CP)-induced genotoxicity through modulation of hepatic ABC transporters. Daily oral administration of E. vesicaria fresh leaves juice (1.0, 1.4 and 2.0 g/kg) for 14 days did not alter genotoxicity biomarkers —alkaline comet assay and micronucleus test —in neither male nor female mice. Instead, repeated intake of this cruciferous decreased CP-induced DNA damage dose-dependently and it caused hepatic overexpression of P-glycoprotein (P-gp; 1.4 and 2.0 g/kg) and multidrug resistance protein 2 (MRP2; 2.0 g/kg), but not breast cancer resistance protein (Bcrp). The antigenotoxic effect of E. vesicaria was prevented by 50 mg/kg verapamil (P-gp inhibitor) or 10 mg/kg indomethacin (MRP2 inhibitor). In turn, CP-induced cytotoxicity (10 mM, 24 h) on human hepatoma cells (HepG2/C3A) was significantly reduced by preincubation with E. vesicaria (1.4 mg/ml; 48 h); this effect was absent when CP was coincubated with 35 μM verapamil, 80 μM indomethacin or 10 μM KO-143 (BCRP inhibitor).Altogether, these results allow us to demonstrate that repeated intake of E. vesicaria exhibited antigenotoxicity, at least in part, by induction of hepatic ABC transporters in vivo in mice as well as in vitro in human liver cells. This could account for other diet-drug interactions.

Morin hydrate ameliorates cisplatin-induced ER stress, inflammation and autophagy in HEK-293 cells and mice kidney via PARP-1 regulation

The present study assessed the possible therapeutic potential of a natural flavonoid morin hydrate (MH), against cisplatin (CP) induced toxicity in HEK-293 cells and mice kidney. Herein, we observed that exposure of HEK-293 cells to CP (20 μM, 24 h) reduced the cell viability, and increased the intracellular ROS generation, nuclear DNA damage, Ca++ release, and accumulation of acidic vacuoles. Concomitantly, acute exposure of CP (30 mg/kg, 72 h) to male ICR mice induced histopathological changes in kidney tissue, and alterations in serum creatinine and blood urea nitrogen (BUN) levels. Oxidative stress mediated ER-stress was evidenced by the reduced expression of antioxidant enzymes such as SOD-1, SOD-2, GR, and Trx, and increased expression levels of CytP450, IRE1-α, PERK, and CHOP. The expression levels of major inflammatory response markers such as NF-κB, TNF-α, IL-1β, COX-2 and iNOS were significantly increased in the HEK-293 cells and mice kidney. Temporal up-regulation of p-AMPK and LC3I/II, and down regulation of mTOR was also noticed after CP treatment. CP-induced DNA damage led to activation of PARP-1, which plays a crucial role in inflammation, apoptosis and autophagy activation. Concurrently, co-treatment of CP-MH and CP-ANI (PARP-1 inhibitor) significantly attenuated the expression level of PARP-1, reduced cellular death, alleviated inflammatory responses, and inhibited autophagy stimulation in HEK-293 cells and mice kidney. On the basis of above findings, we suggest MH as a potential therapeutic agent against CP-induced nephrotoxicity.

Nano-Se attenuates cyclophosphamide-induced pulmonary injury through modulation of oxidative stress and DNA damage in Swiss albino mice.

Chemotherapy is an integral part of modern day treatment regimen but anticancer drugs fail to demarcate between cancerous and normal cells thereby causing severe form of systemic toxicity. Among which pulmonary toxicity is a dreadful complication developed in cancer patients upon cyclophosphamide (CP) therapy. Oxidative stress, fibrosis, and apoptosis are the major patho-mechanisms involved in CP-induced pulmonary toxicity. In the present study, we have synthesized Nano-Se, nanotechnology-based new form of elemental selenium which has significantly lower toxicity and acceptable bioavailability. In order to meet the need of effective drugs against CP-induced adverse effects, nano selenium (Nano-Se) was tested for its possible protective efficacy on CP-induced pulmonary toxicity and bone marrow toxicity. CP intoxication resulted in structural and functional lung impairment which was revealed by massive histopathological changes. Lung injury was associated with oxidative stress/lipid peroxidation as evident by increased in reactive oxygen species, nitric oxide level, and malondialdehyde (MDA) formation with decreased in level of antioxidants such as reduced glutathione, glutathione-S-transferase, glutathione peroxidase, superoxide dismutase, and catalase. Furthermore, CP at a dose of 25 mg/kg b.w. increased pulmonary DNA damage ('comet tail') and triggered DNA fragmentation and apoptosis in mouse bone marrow cells. On the other hand, Nano-Se at a dose of 2 mg Se/kg b.w., significantly inhibited CP-induced DNA damage in bronchoalveolar lavage cells, and decreased the apoptosis and percentage of DNA fragmentation in bone marrow cells and also antagonized the reduction of the activities of antioxidant enzymes and the increase level of MDA. Thus, our results suggest that Nano-Se in pre- and co-administration may serve as a promising preventive strategy against CP-induced pulmonary toxicity.

Intervention in cyclophosphamide induced oxidative stress and DNA damage by a flavonyl-thiazolidinedione based organoselenocyanate and evaluation of its efficacy during adjuvant therapy in tumor bearing mice

A novel flavonyl-thiazolidinedione based organoselenocyanate compound was synthesized and established as nontoxic at the doses of 2.5 and 5 mg/kg b.w. in mice. Oral administration of the compound in combination with cyclophosphamide (CP) resulted in an improved therapeutic efficacy which was mostly evidenced in terms of tumor burden and protection of normal cells. The adjuvant therapy was proved to be immensely significant in increasing the mean survivability of the tumor bearing hosts. Reduction in the tumor volume was manifested through the induction of apoptosis and generation of ROS in transformed cells. Moreover, the organoselenium compound could efficiently suppress CP-induced DNA damage, chromosomal aberration, hepatic damage and enhanced the activities of various antioxidant enzymes in normal cells.

Evaluation of the Bronchorelaxant, Genotoxic, and Antigenotoxic Effects ofCassia alataL.

Aqueous-ethanolic extract of Cassia alata (AECal) and its derived fractions obtained through liquid-liquid fractionation were evaluated for their bronchorelaxant, genotoxic, and antigenotoxic effects. Contractile activity of rats' tracheas in the presence of tested materials, as well as its modifications with different inhibitors and blockers, was isometrically recorded. The antigenotoxic potential of AECal was evaluated on cyclophosphamide- (CP-) induced genotoxicity in the rat. Animals were pretreated with the extract, then liver comet assay was performed. AECal and its chloroformic fractions (CF-AECal) relaxed the contraction induced by Ach, but both were significantly less potent in inhibiting contraction induced by KCl (30 mM; 80 mM). Propranolol, indomethacin, L-NAME, methylene blue, and glibenclamide did not modify the relaxant effect of CF-AECal. TEA altered the response of trachea to CF-AECal. CF-AECal caused a rightward shift without affecting the E max in cumulative concentration-response curves of Ach only at low concentrations. In animals pretreated with the extract, the percentage of CP-induced DNA damage decreased. Our results suggest that (1) muscarinic receptors contribute at least in part to the relaxant effects of CF-AECal; (2) CF-AECal interferes with membrane polarization; and (3) AECal is not genotoxic in vivo and contains chemopreventive phytoconstituents offering protection against CP-induced genotoxicity.

Dose-response assessment of four genotoxic chemicals in a combined mouse and rat micronucleus (MN) and Comet assay protocol

The in vivo micronucleus (MN) assay has proven to be an effective measure of genotoxicity potential. However, sampling a single tissue (bone marrow) for a single indicator of genetic damage using the MN assay provides a limited genotoxicity profile. The in vivo alkaline (pH >13) Comet assay, which detects a broad spectrum of DNA damage, can be applied to a variety of rodent tissues following administration of test agents. To determine if the Comet assay is a useful supplement to the in vivo MN assay, a combined test protocol (MN/Comet assay) was conducted in male B6C3F1 mice and F344/N rats using four model genotoxicants: ethyl methanesulfonate (EMS), acrylamide (ACM), cyclophosphamide (CP), and vincristine sulfate (VS). Test compounds were administered on 4 consecutive days at 24-hr intervals (VS was administered to rats for 3 days); animals were euthanized 4 hr after the last administration. All compounds induced significant increases in micronucleated reticulocytes (MN-RET) in the peripheral blood of mice, and all but ACM induced MN-RET in rats. EMS and ACM induced significant increases in DNA damage, measured by the Comet assay, in multiple tissues of mice and rats. CP-induced DNA damage was detected in leukocytes and duodenum cells. VS, a spindle fiber disrupting agent, was negative in the Comet assay. Based on these results, the MN/Comet assay holds promise for providing more comprehensive assessments of potential genotoxicants, and the National Toxicology Program (NTP) is presently using this combined protocol in its overall evaluation of the genotoxicity of substances of public health concern.

Arsenate and dimethylarsinic acid in drinking water did not affect DNA damage repair in urinary bladder transitional cells or micronuclei in bone marrow

Arsenic is a human skin, lung, and urinary bladder carcinogen, and may act as a cocarcinogen in the skin and urinary bladder. Possible modes of action of arsenic carcinogenesis/cocarcinogenesis include oxidative stress induction and inhibition of DNA damage repair. We investigated the effects of arsenic in drinking water on DNA damage repair in urinary bladder transitional cells and on micronucleus formation in bone marrow. F344 rats were given 100 ppm arsenate [As(V)] or dimethylarsinic acid [DMA(V)] in drinking water for 1 week. The in vivo repair of cyclophosphamide (CP)-induced DNA damage resulting from a single oral gavage of CP, and the in vitro repair of hydrogen peroxide (H(2)O(2))- or formaldehyde-induced DNA damage, resulting from adding H(2)O(2) or formaldehyde into cell medium, were measured by the Comet assay. DMA(V) effects were not observed on either CP-induced DNA damage induction or on DNA repair. Neither DMA(V) nor As(V) increased the H(2)O(2)- or formaldehyde-induced DNA damage, and neither inhibited the repair of H(2)O(2)-induced DNA damage. Neither DMA(V) nor As(V) increased the micronucleus frequency, nor did they elevate micronucleus frequency resulting from CP treatment above the level observed by the treatment with CP alone. These results suggest that arsenic carcinogenesis/cocarcinogenesis in the urinary bladder may not be via DNA damage repair inhibition. To our knowledge this is the first report of arsenic effects on DNA damage repair in the urinary bladder.

Protective effects of ginsenoside Rg3 against cyclophosphamide-induced DNA damage and cell apoptosis in mice

Despite the significant anti-tumor activities, cyclophosphamide (CP) also shows cytotoxicity to normal cells. In order to explore the protective effects of drugs against CP-induced adverse effects, 20(S)-ginsenoside Rg(3) was tested for its possibly protective activities on CP-induced DNA damage and cell apoptosis in mouse bone marrow cells or peripheral lymphocyte cells. In the current study, the alkaline single cell gel electrophoresis (comet assay), flow cytometry assay with annexin V-FITC/PI and AO/EB staining assay were employed to measure DNA strand breakage and cell apoptosis, respectively. The activities of SOD and GPx and the contents of MDA were also tested by the various colormetric methods. The results showed that CP at a dose of 100 mg/kg, i.p. significantly caused DNA damages in both mouse bone marrow cells and peripheral lymphocyte cells, and markedly inhibited the activities of GPx and SOD and increased MDA contents in mouse blood. Moreover, CP at a dose of 200 mg/kg, i.p. triggered apoptosis in mouse bone marrow cells. On the other hand, 20(S)-ginsenoside Rg(3) orally administered at a dose of 20 mg/kg to the animals once a day for 2 days significantly inhibited CP-induced DNA damages in mouse bone marrow cells and peripheral lymphocyte cells, decrease the apoptotic numbers of bone marrow cells, antagonized the reduction of the activities of SOD and GPx, and the increase in MDA contents. In conclusion, 20(S)-ginsenoside Rg(3) showed the significant protective effects on CP-induced cell DNA damage and apoptosis. These effects might be partially attributed to its protective actions against CP-induced oxidative stress.

EVALUATION OF GENETIC TOXICITY FROM ENVIRONMENTAL POLLUTANTS IN DAPHNIA MAGNA AND CHIRONOMUS TENTANS FOR APPLICATION IN ECOLOGICAL RISK ASSESSMENT

The genetic toxicity of environmental pollutants, namely, nonylphenol (NP), bisphenol A (BPA) and chloropyriphos (CP) was investigated in aquatic sentinel species, freshwater crustacean, Daphnia magna, and larva of aquatic midge, Chironomus tentans, using Comet assay. Physiological effect of such pollutants was also investigated by studying the specimens' rates of reproduction, growth and survival. Acute toxicity results showed that, as expected, Daphnia was more sensitive than Chironomus to chemical exposure. The order of acute toxicity was CP > NP > BPA in D. magna and NP > CP > BPA in C. tentans. BPA may exert a genotoxic effect on D. magna and C. tentans, given that DNA strand breaks increased in both species exposed to this compound, whereas NP- and CP-induced DNA damage occurred only in C. tentans. In vivo genotoxic data obtained in aquatic sentinel species could provide valuable information for freshwater quality monitoring. The experiments with NP-exposed D. magna showed that the pollutant has long-term effects on reproduction, whereas no short-term effect on DNA integrity was found, being an example of a false-negative result from the biomarkers perspective. This result could be interpreted that other mechanism than genetic alteration might be involved in NP-induced reproduction failure in D. magna. False-positive results from the genotoxic biomarker obtained in BPA-exposed D. magna and in NP-exposed C. tentans make it difficult to use DNA integrity as an early warning biomarker. However, as the mere presence of genotoxic compounds, which are potentially carcinogenic, is of high concern to human and ecosystem health, it could also be important to rapidly and effectively detect genotoxic compounds in the aquatic system in ways that do not necessarily accompany a higher level of alteration. Considering the potential of D. magna and C. tentans as bioindicator species, and the importance of genotoxic biomarkers in ecotoxicity monitoring, DNA damage in these species could provide useful information for environmental risk assessment.

Modulation of cisplatinum cytotoxicity by p53: effect of p53-mediated apoptosis and DNA repair.

A stable transfectant (S2SN7) of p53-null SaOS-2 (human osteosarcoma) cells expressing wild-type p53 under the tight control of a tetracycline-responsive promoter was used to study the functional roles of p53 in cellular response to cisplatinum (CP). When cells were grown in media containing normal concentrations (10%) of serum, induction of p53 by tetracycline withdrawal resulted in an 8-fold decrease in sensitivity to CP. In contrast, when cells were grown in lower serum (1%) media, induction of p53 led to a 10-fold increase in sensitivity to CP. The p53-mediated sensitivity to CP under lower serum conditions was attributed, at least in part, to increased susceptibility of p53-mediated apoptosis. Under lower serum (0.1-1%) but not normal serum conditions, p53 induction correlated with selective down-regulation of bcl-2, an inhibitor of apoptosis. In addition, a host-cell reactivation assay showed that induction of p53 caused a significant increase in repair of CP-induced DNA damage under normal serum but not low serum conditions. These data suggest that growth conditions may modulate and possibly reverse p53-mediated CP sensitivity by altering p53-mediated gene regulation and, as a result, susceptibility to apoptosis. They also suggest that a combined effect of p53-mediated apoptosis and DNA repair may be the ultimate determinant in p53-mediated cellular resistance or sensitivity to chemotherapeutic drugs.