Effect of styrene oxide and diethyl maleate on expression of cytochrome P450 family 1 and glutathione store in mouse liver

Purpose: To determine the effect of the glutathione (GSH) suppressors styrene oxide (SO) and diethyl maleate (DEM) on the hepatic expression of cytochrome P450 family 1 (Cyp1) isoforms that are related to carcinogenesis including Cyp1a1, Cyp1a2, and Cyp1b1. Methods: Seven-week-old ICR mice were intraperitoneally injected with SO (150 and 300 mg/kg/day), DEM (175 and 350 mg/kg/day), or N-acetylcysteine (NAC; 300 and 600 mg/kg/day) for 7, 14, or 28 days. Plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, hepatic Cyp1 expression, total glutathione, reduced glutathione (GSH), and oxidized glutathione (GSSG) were determined. Results: ALT and AST levels were markedly increased by SO and DEM while GSH/GSSG ratio was decreased by SO in all treatments (p < 0.05), while high dose (350 mg/kg/day) DEM significantly suppressed GSH/GSSG ratio at 28 days (p < 0.05). The expressions of Cyp1a1, Cyp1a2, and Cyp1b1 were induced by SO and DEM, corresponding with induction of ethoxy/methoxy-resorufin O- dealkylase activities. Conclusion: The Cyp1 family metabolizes procarcinogens to carcinogenic DNA adducts; exposure to the industrial solvents, SO and DEM, raises the risk of carcinogenesis via GSH depletion coupled with Cyp1 induction.

Feeding Brassica vegetables to rats leads to the formation of characteristic DNA adducts (from 1-methoxy-3-indolylmethyl glucosinolate) in many tissues

Juices of Brassica vegetables are mutagenic and form characteristic DNA adducts in bacteria and mammalian cells. In this study, we examined whether such adducts are also formed in vivo in animal models. Rats fed raw broccoli ad libitum in addition to normal laboratory chow for 5 weeks showed one major adduct spot and sometimes an additional minor adduct spot in liver, kidney, lung, blood and the gastrointestinal tract, as determined by 32P-postlabelling/thin-layer chromatography. Adducts with the same chromatographic properties were formed when herring sperm DNA (or dG-3’-phosphate) was incubated with 1-methoxy-3-indolylmethyl glucosinolate (phytochemical present in Brassica plants), in the presence of myrosinase (plant enzyme that hydrolyses glucosinolates to bioactive breakdown products). UPLC–MS/MS analysis corroborated this finding: 1-Methoxy-3-indolylmethyl-substituted purine nucleosides were detected in the hepatic DNA of broccoli-fed animals, but not in control animals. Feeding raw cauliflower led to the formation of the same adducts. When steamed rather than raw broccoli was used, the adduct levels were essentially unchanged in liver and jejunum, but elevated in large intestine. Due to inactivation of myrosinase by the steaming, higher levels of the glucosinolates may have reached the large bowl to be activated by glucosidases from intestinal bacteria. In conclusion, the consumption of common Brassica vegetables can lead to the formation of substantial levels of DNA adducts in animal models. The adducts can be attributed to a specific phytochemical, neoglucobrassicin (1-methoxy-3-indolylmethyl glucosinolate).

Dietary Resveratrol Alleviates AFB1-Induced Ileum Damage in Ducks via the Nrf2 and NF-κB/NLRP3 Signaling Pathways and CYP1A1/2 Expressions

The aim of this study was to explore the mechanism underlying the protective effects of resveratrol against Aflatoxin B1-induced ileum injury in ducks. A corn–soybean meal-basal diet and two test diets (500 mg/kg resveratrol +0.2 mg Aflatoxin B1/kg, 0.2 mg AFB1/kg) were used in a 10-wk design trial (n = 15 ducks/group). These results showed that the toxicity of Aflatoxin B1 significantly reduced the antioxidant capacity of duck ileum and induced inflammation, oxidative stress, mitochondrial dysfunction and DNA damage in ducks. The expression of genes, including CYP1A2, CYP2A6, and CYP3A4, at the mRNA level was significantly upregulated (p < 0.05) by AFB1. The level of Nrf2 was suppressed (p < 0.05) and the mRNA and protein level of NF-κB was activated (p < 0.05) in the AFB1 group. However, supplementation with 500 mg/kg dietary resveratrol in Aflatoxin B1-induced ducks significantly ameliorated these alterations and decreased the mRNA expression of CYP1A1 and CYP1A2 (p < 0.05) and the production of AFB1-DNA adducts (p < 0.05). The results proved that resveratrol alleviated ileum injury induced by AFB1, decreased the production of AFB1-DNA adducts by downregulating the expression of CYP1A1 and CYP1A2, and reduced DNA damage and oxidative stress via the Nrf2/ Keap1 and NF-κB/NLRP3 signaling pathways.

Current and Future Methodology for Quantitation and Site-specific Mapping the Location of DNA Adducts

Formation of DNA adducts is a key event for a genotoxic mode of action and its formation is often use as surrogate for mutation and cancer. Interest in DNA adducts are twofold, first, to demonstrate exposure, and second, to link DNA adduct location to subsequent mutations or altered gene regulation. High chemically specific mass spectrometry methods have been established for DNA adduct quantitation and elegant bio-analytic methods utilizing enzymes, various chemistries, and molecular biology methods to visualize the location of DNA adducts. Traditionally, these highly specific methods cannot be combined, and the results are incomparable. Initially developed for single-molecule DNA sequencing, nanopore-type technologies are expected to enable simultaneous quantitation and location of DNA adducts across the genome. We will briefly summarize the current methodologies for state-of-the-art quantitation of DNA adduct levels and mapping of DNA adducts and describe novel single-molecule DNA sequencing technology that is expected to achieve both measures simultaneously. Emerging technologies are expected to soon provide a comprehensive picture of the exposome and identify gene regions susceptible to DNA adduct formation.

Dietary Curcumin Alleviated Aflatoxin B1-Induced Acute Liver Damage in Ducks by Regulating NLRP3–Caspase-1 Signaling Pathways

Aflatoxin B1 (AFB1) is a mycotoxin widely distributed in animal feed and human food; it represents a serious threat to human and animal health. This study investigates the mechanism by which dietary curcumin protected liver against acute damage caused by AFB1 administration in ducks. One-day-old male ducks (n = 450) were randomly assigned to three groups, the control group, the AFB1 group, and the AFB1 + curcumin group; the first group were fed with basic diet, while the third group was fed basic diet containing 500 mg/kg curcumin. Ducks in the AFB1 group and AFB1 + curcumin group were challenged with AFB1 at the age of 70 days. The results show that AFB1 administration caused liver damage, increased CYP450 content and AFB1-DNA adducts in the liver, and induced oxidative stress and inflammatory response in the liver. Dietary curcumin significantly inhibited the generation of H2O2 and MDA in liver, activated the Nrf2-ARE signaling pathway, and suppressed the NLRP3–caspase-1 signaling pathway in the liver of ducks. Conclusively, curcumin in diet could protect duck liver against the generation of AFB1-DNA adducts, toxicity, oxidation stress and inflammatory response induced by AFB1 through regulating the NLRP3–caspase-1 signaling pathways, demonstrating that curcumin is a potential feed additive agent to reduce the serious harmful effects of AFB1 on duck breeding.

A DNA adductome analysis revealed a reduction in the global level of C5-hydroxymethyl-2′-deoxycytidine in the non-tumoral upper urinary tract mucosa of urothelial carcinoma patients

Background DNA adducts, covalent modifications to DNA due to exposure to specific carcinogens, cause the mispairing of DNA bases, which ultimately results in DNA mutations. DNA methylation in the promoter region, another type of DNA base modification, alters the DNA transcription process, and has been implicated in carcinogenesis in humans due to the down-regulation of tumor suppressor genes. Difficulties are associated with demonstrating the existence of DNA adducts or chemically modified bases in the human urological system. Apart from aristolochic acid-DNA adducts, which cause urothelial carcinoma and endemic nephropathy in a particular geographical area (Balkan), limited information is currently available on DNA adduct profiles in renal cell carcinoma and upper urinary tract urothelial carcinoma, including renal pelvic cancer and ureteral cancer. Method To elucidate the significance of DNA adducts in carcinogenesis in the urothelial system, we investigated 53 DNA adducts in the non-tumoral renal parenchyma and non-tumoral renal pelvis of patients with renal cell carcinoma, upper urinary tract urothelial carcinoma, and other diseases using liquid chromatography coupled with tandem mass spectrometry. A comparative analysis of tissue types, the status of malignancy, and clinical characteristics, including lifestyle factors, was performed. Results C5-Methyl-2′-deoxycytidine, C5-hydroxymethyl-2′-deoxycytidine (5hmdC), C5-formyl-2′-deoxycytidine, 2′-deoxyinosine, C8-oxo-2′-deoxyadenosine, and C8-oxo-2′-deoxyguanosine (8-OHdG) were detected in the renal parenchyma and renal pelvis. 8-OHdG was more frequently detected in the renal pelvis than in the renal cortex and medulla (p = 0.048 and p = 0.038, respectively). 5hmdC levels were significantly lower in the renal pelvis of urothelial carcinoma patients (n = 10) than in the urothelium of patients without urothelial carcinoma (n = 15) (p = 0.010). Regarding 5hmdC levels in the renal cortex and medulla, Spearman’s rank correlation test revealed a negative correlation between age and 5hmdC levels (r = − 0.46, p = 0.018 and r = − 0.45, p = 0.042, respectively). Conclusions The present results revealed a reduction of 5hmdC levels in the non-tumoral urinary tract mucosa of patients with upper urinary tract urothelial carcinoma. Therefore, the urothelial cell epithelia of patients with upper urinary tract cancer, even in non-cancerous areas, may be predisposed to urothelial cancer.

Identification of new candidate biomarkers to support doxorubicin treatments in canine cancer patients

Background Both human and veterinary cancer chemotherapy are undergoing a paradigm shift from a “one size fits all” approach to more personalized, patient-oriented treatment strategies. Personalized chemotherapy is dependent on the identification and validation of biomarkers that can predict treatment outcome and/or risk of toxicity. Many cytotoxic chemotherapy agents, including doxorubicin, base their mechanism of action by interaction with DNA and disruption of normal cellular processes. We developed a high-resolution/accurate-mass liquid chromatography-mass spectrometry DNA screening approach for monitoring doxorubicin-induced DNA modifications (adducts) in vitro and in vivo. We used, for the first time, a new strategy involving the use of isotope-labeled DNA, which greatly facilitates adduct discovery. The overall goal of this work was to identify doxorubicin-DNA adducts to be used as biomarkers to predict drug efficacy for use in veterinary oncology. Results We used our novel mass spectrometry approach to screen for adducts in purified DNA exposed to doxorubicin. This initial in vitro screening identified nine potential doxorubicin-DNA adduct masses, as well as an intense signal corresponding to DNA-intercalated doxorubicin. Two of the adduct masses, together with doxorubicin and its metabolite doxorubicinol, were subsequently detected in vivo in liver DNA extracted from mice exposed to doxorubicin. Finally, the presence of these adducts and analytes was explored in the DNA isolated from dogs undergoing treatment with doxorubicin. The previously identified nine DOX-DNA adducts were not detected in these preliminary three samples collected seven days post-treatment, however intercalated doxorubicin and doxorubicinol were detected. Conclusions This work sets the stage for future evaluation of doxorubicin-DNA adducts and doxorubicin-related molecules as candidate biomarkers to personalize chemotherapy protocols for canine cancer patients. It demonstrates our ability to combine in one method the analysis of DNA adducts and DNA-intercalated doxorubicin and doxorubicinol. The last two analytes interestingly, were persistent in samples from canine patients undergoing doxorubicin chemotherapy seven days after treatment. The presence of doxorubicin in all samples suggests a role for it as a promising biomarker for use in veterinary chemotherapy. Future studies will involve the analysis of more samples from canine cancer patients to elucidate optimal timepoints for monitoring intercalated doxorubicin and doxorubicin-DNA adducts and the correlation of these markers with therapy outcome.