Deconvolution of Systemic Pharmacokinetics Predicts Inhaled Aerosol Dosimetry of Nicotine.

Absorption of inhaled compounds can occur from multiple sites based on upper and lower respiratory tract deposition, and clearance mechanisms leading to differential local and systemic pharmacokinetics. Deriving inhaled aerosol dosimetry and local tissue concentrations for nose-only exposure in rodents and inhaled products in humans is challenging. In this study we use inhaled nicotine as an example to identify regional respiratory tract deposition, absorption fractions, and their contribution toward systemic pharmacokinetics in rodents and humans. A physiologically based pharmacokinetic (PBPK) model was constructed to describe the disposition of nicotine and its major metabolite, cotinine. The model description for the lungs was simplified to include an upper respiratory tract region with active mucociliary clearance and a lower respiratory tract region. The PBPK model parameters such as rate of oral absorption, metabolism and clearance were fitted to the published nicotine and cotinine plasma concentrations post systemic administration and oral dosing. The fractional deposition of inhaled aerosol in the upper and lower respiratory tract regions was estimated by fitting the plasma concentrations. The model predicted upper respiratory tract deposition was 63.9% for nose-only exposure to nicotine containing nebulized aqueous aerosol in rats and 60.2% for orally inhaled electronic vapor product in humans. A marked absorption of nicotine from the upper respiratory tract and the gastrointestinal tract for inhaled aqueous aerosol contributed to the differential systemic pharmacokinetics in rats and humans. The PBPK model derived dosimetry shows that the current aerosol dosimetry models with their posteriori application using independent aerosol physicochemical characterization to predict aerosol deposition are insufficient and will need to consider complex interplay of inhaled aerosol evolutionary process. While the study highlights the needs for future research, it provides a preliminary framework for interpreting pharmacokinetics of inhaled aerosols to facilitate the analysis of in vivo exposure-responses for pharmacological and toxicological assessments.

In vitro testing of salt coating of fabrics as a potential antiviral agent in reusable face masks.

During the coronavirus disease (COVID-19) pandemic, wearing face masks in public spaces became mandatory in most countries. The risk of self-contamination when handling face masks, which was one of the earliest concerns, can be mitigated by adding antiviral coatings to the masks. In the present study, we evaluated the antiviral effectiveness of sodium chloride deposited on a fabric suitable for the manufacturing of reusable cloth masks using techniques adapted to the home environment. We tested eight coating conditions, involving both spraying and dipping methods and three salt dilutions. Influenza A H3N2 virus particles were incubated directly on the salt-coated materials, collected, and added to human 3D airway epithelial cultures. Live virus replication in the epithelia was quantified over time in collected apical washes. Relative to the non-coated material, salt deposits at or above 4.3 mg/cm2 markedly reduced viral replication. However, even for larger quantities of salt, the effectiveness of the coating remained dependent on the crystal size and distribution, which in turn depended on the coating technique. These findings confirm the suitability of salt coating as antiviral protection on cloth masks, but also emphasize that particular attention should be paid to the coating protocol when developing consumer solutions.

Evaluation of toxicity of aerosols from flavored e-liquids in Sprague-Dawley rats in a 90-day OECD inhalation study, complemented by transcriptomics analysis.

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

Multi-omics systems toxicology study of mouse lung assessing the effects of aerosols from two heat-not-burn tobacco products and cigarette smoke.

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE-/- mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2-a potential and a candidate MRTP based on the heat-not-burn (HnB) principle-compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme-Hmox-bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice.

A 90-day OECD TG 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of the aerosol from the carbon heated tobacco product version 1.2 (CHTP1.2) compared with cigarette smoke. II. Systems toxicology assessment.

Modified risk tobacco products (MRTPs) have the potential to reduce smoking-related health risks. The Carbon Heated Tobacco Product 1.2 (CHTP1.2) is a potential MRTP that uses a pressed carbon heat source to generate an aerosol by heating tobacco. Here, we report the results from the systems toxicology arm of a 90-day rat inhalation study (OECD test guideline 413) to assess the effects of CHTP1.2 aerosol compared with cigarette smoke (CS). Transcriptomics, proteomics, and lipidomics analyses complemented the standard endpoints. In the respiratory nasal epithelium, CS induced an adaptive tissue and inflammatory response, which was much weaker after CHTP1.2 aerosol exposure, mostly limited to the highest CHTP1.2 concentration (at twice the 3R4F CS concentration: 50 vs. 23 µg nicotine/L), in female rats. In the lungs, the effects of CS exposure included inflammatory and cellular stress responses, which were absent or much lower after CHTP1.2 aerosol exposure. Outside of the respiratory tract, CS and CHTP1.2 aerosol induced effects that were previously associated with exposure to any nicotine-containing aerosol, e.g., lower lipid concentrations in serum. Overall, this systems toxicology analysis complements and confirms the results from classical toxicological endpoints and further suggests potentially reduced respiratory health risks of CHTP1.2.

Toxicity of the main electronic cigarette components, propylene glycol, glycerin, and nicotine, in Sprague-Dawley rats in a 90-day OECD inhalation study complemented by molecular endpoints.

While the toxicity of the main constituents of electronic cigarette (ECIG) liquids, nicotine, propylene glycol (PG), and vegetable glycerin (VG), has been assessed individually in separate studies, limited data on the inhalation toxicity of them is available when in mixtures. In this 90-day subchronic inhalation study, Sprague-Dawley rats were nose-only exposed to filtered air, nebulized vehicle (saline), or three concentrations of PG/VG mixtures, with and without nicotine. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared with vehicle exposure, the PG/VG aerosols showed only very limited biological effects with no signs of toxicity. Addition of nicotine to the PG/VG aerosols resulted in effects in line with nicotine effects observed in previous studies, including up-regulation of xenobiotic enzymes (Cyp1a1/Fmo3) in the lung and metabolic effects, such as reduced serum lipid concentrations and expression changes of hepatic metabolic enzymes. No toxicologically relevant effects of PG/VG aerosols (up to 1.520  mg PG/L + 1.890 mg VG/L) were observed, and no adverse effects for PG/VG/nicotine were observed up to 438/544/6.6 mg/kg/day. This study demonstrates how complementary systems toxicology analyses can reveal, even in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine.

Evaluation of the Tobacco Heating System 2.2. Part 7: Systems toxicological assessment of a mentholated version revealed reduced cellular and molecular exposure effects compared with mentholated and non-mentholated cigarette smoke.

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M.

Evaluation of the Tobacco Heating System 2.2. Part 6: 90-day OECD 413 rat inhalation study with systems toxicology endpoints demonstrates reduced exposure effects of a mentholated version compared with mentholated and non-mentholated cigarette smoke.

The toxicity of a mentholated version of the Tobacco Heating System (THS2.2M), a candidate modified risk tobacco product (MRTP), was characterized in a 90-day OECD inhalation study. Differential gene and protein expression analysis of nasal epithelium and lung tissue was also performed to record exposure effects at the molecular level. Rats were exposed to filtered air (sham), to THS2.2M (at 15, 23 and 50 µg nicotine/l), to two mentholated reference cigarettes (MRC) (at 23 µg nicotine/l), or to the 3R4F reference cigarette (at 23 µg nicotine/l). MRCs were designed to meet 3R4F specifications. Test atmosphere analyses demonstrated that aldehydes were reduced by 75%-90% and carbon monoxide by 98% in THS2.2M aerosol compared with MRC smoke; aerosol uptake was confirmed by carboxyhemoglobin and menthol concentrations in blood, and by the quantities of urinary nicotine metabolites. Systemic toxicity and alterations in the respiratory tract were significantly lower in THS2.2M-exposed rats compared with MRC and 3R4F. Pulmonary inflammation and the magnitude of the changes in gene and protein expression were also dramatically lower after THS2.2M exposure compared with MRCs and 3R4F. No menthol-related effects were observed after MRC mainstream smoke-exposure compared with 3R4F.

Mechanism of an indirect effect of aqueous cigarette smoke extract on the adhesion of monocytic cells to endothelial cells in an in vitro assay revealed by transcriptomics analysis.

The adhesion of monocytic cells to the "dysfunctional" endothelium constitutes a critical step in the initiation of atherosclerosis. Cigarette smoke (CS) has been shown to contribute to this process, the complex mechanism of which still needs to be unraveled. We developed an in vitro adhesion assay to investigate the CS-induced adhesion of monocytic MM6 cells to human umbilical vein endothelial cells (HUVECs) following exposure to an aqueous CS extract (smoke-bubbled phosphate buffered saline: sbPBS), reasoning that in vivo monocytes and endothelial cells are exposed primarily to soluble constituents from inhaled CS absorbed through the lung alveolar wall. MM6 cell adhesion was increased exclusively by the conditioned medium from sbPBS-exposed MM6 cells, not by direct sbPBS exposure of the HUVECs within a range of sbPBS doses. Using a transcriptomics approach followed by confirmation experiments, we identified different exposure effects on both cell types and a key mechanism by which sbPBS promoted the adhesion of MM6 cells to HUVECs. While sbPBS provoked a strong oxidative stress response in both cell types, the expression of E-selectin, VCAM-1 and ICAM-1, responsible for the adhesion of MM6 cells to HUVECs, was induced in the latter through a proinflammatory paracrine effect. We confirmed that this effect was driven mainly by TNFα produced by MM6 cells exposed to sbPBS. In conclusion, we have elucidated an indirect mechanism by which sbPBS increases the adhesion of monocytic cells to endothelial cells in this in vitro assay that was designed for tobacco product risk assessment while mimicking the in vivo exposure conditions as closely as possible.

Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation.

Cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD) and a risk factor for both lung and cardiovascular (CV) diseases, which are rarely investigated concomitantly. Although smoking cessation shows clear CV risk benefit, lung-related disease risk remains higher in former smokers than in never smokers. We sought to determine the differential molecular responses of murine respiratory tissues to better understand the toxicity pathways involved in smoking-related disease risk and those related to the benefits of smoking cessation. ApoE(-/-) mice were exposed to mainstream cigarette smoke (CS) or a smoking cessation-mimicking protocol for up to 6 months and transcriptomics analysis of nasal epithelium and lung parenchyma performed. We supported our gene expression profiling approach with standard lung histopathology and bronchoalveolar lavage fluid (BALF) analysis. Many BALF analytes involved in functions ranging from inflammation to cell proliferation and tissue remodeling were found elevated in BALF. Gene expression levels of these molecules were also increased in lung tissue, suggesting that the inflammatory response was the result of local tissue activation and the contribution of recruited inflammatory cells. Gene set enrichment analysis (GSEA) of expression data from murine lungs and nasal epithelium showed distinct activation patterns of inflammation, complement, and xenobiotic metabolism pathways during CS exposure that were deactivated upon smoking cessation. Pathways involved in cell proliferation and tissue remodeling were activated by CS and progressively deactivated upon smoke exposure cessation. Differential CS-mediated responses of pulmonary and nasal tissues reflect common mechanisms but also the varying degrees of epithelial functional specialization and exposure along the respiratory tract.

Cigarette-smoke-induced atherogenic lipid profiles in plasma and vascular tissue of apolipoprotein E-deficient mice are attenuated by smoking cessation.

Tobacco smoke exerts perturbations on lipid metabolism and arterial cell function that accelerate atherosclerosis. Lipidomics has emerged as a key technology in helping to elucidate the lipid-related mechanisms of atherosclerosis. In this study, we investigated the effects of smoking cessation on plaque development and aortic arch content of various lipid molecular classes and species. Apolipoprotein E-deficient mice were exposed to fresh air (sham) or to mainstream cigarette smoke (CS) for 6 months, or to CS for 3 months followed by sham for 3 months (cessation group). Lipids from plasma and aortic arches, plasma lipoprotein profiles and plaque morphometry measurements were analyzed. We already showed that CS exposure accelerated plaque size and total cholesterol content of the aortic arch at 3 and 6 months. Marked increases were seen in the relative enrichment of cholesteryl esters, phospholipids, sphingomyelins, and glycosphingolipids. Smoking cessation slowed plaque progression and resulted in lower levels of many lipid species in plasma and aortic arch. While CS exposure promoted rapid lipid accumulation in mouse aorta, smoking cessation translated into a slow removal of lipids from the vessel wall. Despite the smoking cessation-dependent metabolic changes leading to increased animal body weight, accumulation of proatherogenic lipids in the vessel was halted after exposure cessation, indicating that the clinical benefits of smoking cessation translate directly to the vessel wall and its lipid makeup.

Modulation of atherogenic lipidome by cigarette smoke in apolipoprotein E-deficient mice.

OBJECTIVE: Although relationships between smoking and cardiovascular diseases (CVD), and between CVD and lipids are established, the direct impact of smoking on lipidomes is not well understood. We investigated the effect of mainstream cigarette smoke (CS) exposure on plasma, liver, and aorta molecular lipid profiles, and liver transcriptome in the ApoE(-/-) mouse, a well-established mouse model for human atherogenesis. METHODS: Plasma, liver, and aorta samples from ApoE(-/-) mice exposed to CS or fresh air (sham) for six months were extracted for lipids using robotic-assisted method and analyzed by mass spectrometry. Gene expression in the liver was obtained on microarrays. Development of atherosclerosis in the aorta was further assessed by plaque size in the aortic arch and lipoprotein concentration in plasma and plaque. RESULTS: CS increased most lipid classes and molecular lipid species. In plasma, free cholesterol, ceramides, cerebrosides, and most phospholipids were increased in CS-exposed mice. In the liver, several lipid species including free and esterified cholesterol, triacylglycerols, phospholipids, sphingomyelins, and ceramides were elevated. In the aorta, more than 2-fold higher cholesteryl ester (CE), lysophosphatidylcholine, and glucosyl/galactosylceramide levels were seen. Moreover, CS exposure induced a significant decrease in several plasma CE and phosphatidylcholine species that contained polyunsaturated fatty acids. Genes involved in amino acid and lipid metabolism showed perturbed transcription profiles in the liver. CONCLUSION: We have quantified some of the molecular changes that accompany the increase of plaque size that is accelerated by CS exposure in the aortae of ApoE(-/-) mice. These results suggest that specific changes in the lipidome and transcriptome, for example in ceramide and polyunsaturated fatty acid species, may be associated with atherosclerosis.

Cigarette smoke and LDL cooperate in reducing nitric oxide bioavailability in endothelial cells via effects on both eNOS and NADPH oxidase.

The ubiquitous free radical nitric oxide (NO) plays an important role in many biological processes, including the regulation of both vascular tone and inflammatory response; however, its role in the effects of cigarette smoke exposure on atherosclerosis remains unclear. Our aim was to study the mechanisms of NO regulation in endothelial cells in response to cigarette smoke exposure in vitro. Using human umbilical vein endothelial cells (HUVEC), we have demonstrated that combining non-toxic concentrations of cigarette smoke bubbled through PBS (smoke-bubbled PBS [sbPBS]) with native LDL (nLDL) significantly reduces the amount of bioavailable NO. The effect is comparable to that seen with oxidized LDL (oxLDL), but has not been seen with sbPBS or nLDL alone. Mechanistic investigations showed that the combination of sbPBS+nLDL did not reduce the amount of endothelial nitric oxide synthase (eNOS), but did inhibit its enzymatic activity. Concomitantly, both sbPBS+nLDL and oxLDL significantly increased the production of reactive oxygen species (ROS) in the form of superoxide anions ((·)O(2)(-)) and peroxynitrite (ONOO(-)) in HUVEC. Selective inhibition of NADPH oxidase prevented this response. Incubation of sbPBS+nLDL revealed the formation of 7-ketocholesterol (7-KC) and 7-hydroxycholesterol, which are indicators for oxidative modification of LDL. This could explain the reported increase in circulatory levels of oxLDL in smokers. Our results suggest that reduction of functional NO in response to a combination of sbPBS+nLDL is secondary to both reduction of eNOS activity and stimulation of NADPH oxidase activity. Because sbPBS alone showed no effect on eNOS activity or ROS formation, nLDL should be included in cigarette-smoke-related mechanistic in vitro experiments on endothelial cells to be more reflective of the clinical situation.

Cigarette smoke enhances abdominal aortic aneurysm formation in angiotensin II-treated apolipoprotein E-deficient mice.

Cigarette smoke, hyperlipidemia, and hypertension with the risk of development and progression of atherosclerosis and associated pathologies such as abdominal aortic aneurysm (AAA) are correlated. We examined the interaction of cigarette mainstream smoke (MS) and angiotensin-II (Ang II)-induced hypertension in the atherosclerotic process using hyperlipidemic apolipoprotein E-knockout (ApoE(-/-)) mice. ApoE(-/-) mice were treated with Ang II for 4 weeks and then further exposed to MS or to fresh air for 4 weeks. AAA formation was observed in all mice treated with Ang II, regardless of smoke exposure; however, smoke exposure increased the incidence of AAA in these mice. Ang II treatment resulted in higher gene expression of matrix metalloproteinases (MMP)-2, -3, -8, -9, and -12 in the abdominal aortas, which was further increased by MS exposure. The proteolytic activity of MMP-2 and MMP-9 was also enhanced in Ang II-treated mice exposed to MS, but only minor changes were seen with either smoke exposure or Ang II treatment alone. This study shows for the first time that both formation and severity of AAA in hypertensive ApoE(-/-) mice are accelerated by exposure to MS and that the proteolytic activity of MMPs is enhanced by the combination of Ang II and MS.

Cigarette smoke exposure promotes arterial thrombosis and vessel remodeling after vascular injury in apolipoprotein E-deficient mice.

BACKGROUND: Cigarette smoking is a major risk factor for the development of cardiovascular disease. However, in terms of the vessel wall, the underlying pathomechanisms of cigarette smoking are incompletely understood, partly due to a lack of adequate in vivo models. METHODS: Apolipoprotein E-deficient mice were exposed to filtered air (sham) or to cigarette mainstream smoke at a total particulate matter (TPM) concentration of 600 microg/l for 1, 2, 3, or 4 h, for 5 days/week. After exposure for 10 +/- 1 weeks, arterial thrombosis and neointima formation at the carotid artery were induced using 10% ferric chloride. RESULTS: Mice exposed to mainstream smoke exhibited shortened time to thrombotic occlusion (p < 0.01) and lower vascular patency rates (p < 0.001). Morphometric and immunohistochemical analysis of neointimal lesions demonstrated that mainstream smoke exposure increased the amount of alpha-actin-positive smooth muscle cells (p < 0.05) and dose-dependently increased the intima-to-media ratio (p < 0.05). Additional analysis of smooth muscle cells in vitro suggested that 10 microg TPM/ml increased cell proliferation without affecting viability or apoptosis, whereas higher concentrations (100 and 500 microg TPM/ml) appeared to be cytotoxic. CONCLUSIONS: Taken together, these findings suggest that cigarette smoking promotes arterial thrombosis and modulates the size and composition of neointimal lesions after arterial injury in apolipoprotein E-deficient mice.

Glutathione S-transferase polymorphisms and ochratoxin A toxicity in primary human urothelial cells.

The mycotoxin ochratoxin A (OTA) is a worldwide contaminant of human food. OTA is genotoxic, immunotoxic, teratogenic and carcinogenic in rodents and can cause nephropathy in pigs. High amounts of OTA can cause nephropathy in humans. Moreover, evidence has been accumulated that OTA is a genotoxic carcinogen. Nevertheless, the mechanism that leads to OTA toxicity has not been fully resolved and it is discussed if a bioactivation of OTA is necessary or not. In this study the genotoxicity of OTA was investigated in primary human urothelial cells by means of alkaline single cell gel electrophoresis (Comet assay). Primary cultured human urothelial cells derived from tissue specimens of urological patients were incubated with 100 microM OTA for 3 h. In contrast to recently published results in MDCK cell lines, the cell cultures showed great interindividual differences in the extent of DNA damage. To evaluate these great interindividual differences the influence of the genotype of the isoenzymes of glutathione S-transferase (GST), namely GSTT1, GSTM1 and GSTP1 on the genotoxic potential of OTA was examined. The genotypes of these polymorphic enzymes were determined by polymerase chain reaction (PCR) and the distributions of the genotypes were correlated with the extent of DNA damage. We found associations between the genotypes of the polymorphic GST isoenzymes and the extent of DNA damage between subgroups with and without OTA-related DNA damage. From these results we conclude that genetic predisposition has the potential to influence OTA genotoxicity.

Detection of ochratoxin A-induced DNA damage in MDCK cells by alkaline single cell gel electrophoresis (comet assay).

The mycotoxin ochratoxin A (OTA), a widespread contaminant of food and feedstuffs, is nephrotoxic, immunosuppressive and carcinogenic in domestic and laboratory animals. Additionally, it is suspected as being responsible for urinary tract tumours in patients suffering from Balkan endemic nephropathy. Moreover, evidence has accumulated that OTA is a genotoxic carcinogen, although the mechanism that results in DNA damage has not been fully resolved. In this study, the induction of DNA damage by OTA and the subsequent DNA repair was investigated by alkaline single cell gel electrophoresis (comet assay) in cells originally derived from the kidney, a target organ of OTA. With modifications of the method, the influence of OTA uptake into the cells and of DNA repair on the genotoxic effect of OTA should be investigated. In Madin-Darby canine kidney (MDCK) cells, OTA induced single-strand breaks in a concentration dependent manner. When an external metabolising enzyme system (S9-mix from rat liver) was added, this genotoxic effect was significantly stronger. By co-incubation with methotrexate or with the mycotoxin citrinin, a substrate of the organic anion transporter, the adverse effect of OTA was inhibited. When DNA repair was inhibited by addition of cytosine arabinoside and hydroxyurea, the tail length increased dramatically and all treated cells showed single-strand breaks. A further culture of the damaged cells in the absence of any supplement resulted in a complete repair of the DNA damage within 2 h. Adverse effects on the mechanisms of DNA repair, or exposure to OTA in periods of reduced DNA repair capacity may influence the genotoxic potency of OTA and have to be regarded as a further mechanism by which genotoxic effects of OTA can be performed.

Is there an influence of enzyme polymorphisms on ochratoxin A genotoxicity?

Primary cultured human urothelial cells derived from ureter specimens of urological patients were used to evaluate induction of DNA-damage by OTA in the alkaline single-cell gel electrophoresis (comet) assay. With the cultured cells from each donor a separate comet assay was performed and tail length of the damaged DNA was measured. A broad spectrum of effects was detected between the individual cell cultures with effects reaching from tail lengths on control level up to strongly enhanced tail lengths.All donors of urothelial tissue were additionally genotyped for several xenobiotic metabolising enzymes (cytochrome P450 1A2, glutathione S-transferases T1, M1, and P1, N-acetyltransferase 2) in lymphocyte DNA. The genotype was then correlated with the genotoxic effects obtained in the comet assay.No correlation was found with CYP1A2, GSTT1, and GSTM1 genotypes whereas for GSTP1 stronger genotoxic effects were found in cells from donors with hetero-and homozygously mutated (w/m, m/m) genotypes compared to homozygous wildtypes. The strongest hint for a correlation was found for NAT2, as cells from donors with homozygous mutated alleles (m/m), known as slow acetylators, displayed a higher susceptibility to OTA in the comet assay than cells from donors with the heterozygously mutated or wildtype alleles (rapid acetylators).