Arsenic Genotoxicity Research Articles

Myricetin ameliorates arsenic-induced hematological changes, immune dysfunction, oxidative stress, hepatic and renal injuries and promotes inflammatory genes in rats.

Arsenic (ARS) is a toxic heavy metal that poses a significant concern for both animal and human health. The study investigated the ameliorative effect of myricetin (MRC) against arsenic-induced immune dysfunction, oxidative stress, hematological changes, hepatic and renal injuries, and inflammatory gene expression in rats. Rats were divided into 4 groups: the control group (CON) received orally administered distilled water (1 ml/rat), and the ARS group received 10 mg/kg orally, the MRC group received 5 mg of MRC/kg orally, and the co-treated group (ARS+MRC) received 10 mg/kg of ARS and 5 mg/kg b.w. of MRC orally. The results showed that co-treatment of ARS-exposed rats with MRC significantly corrected erythrocyte parameters (except MCV) and leukocyte parameters (except basophils; p < 0.05). Furthermore, the ARS group significantly reduced total proteins and globulins while significantly increasing liver functions and uric acid levels (p < 0.05). Co-administration with MRC significantly mitigated the heart indices (gamma-glutamyl transferase, creatine phosphokinase, CK, lactate dehydrogenase) and lipid dysfunction caused by ARS exposure (p < 0.05). In ARS-exposed rats, there was a significant reduction in antioxidant enzymes and immunoglobulins (IgG and IgM), as well as significantly increased oxidative stress (p < 0.05). The MRC treatment effectively restored the redox status and immune variables that were disrupted by ARS exposure. Serum levels of nitric acid and lysosome were significantly lower, while levels of IL-4, TNF-α, and IFN-γ were higher in the ARS group compared to the other groups (p < 0.05). Immunohistopathology revealed that the expression of Cox2 in kidney and liver tissues varied from mild to moderate in the ARS+MRC group. Furthermore, the ARS-induced upregulation of mRNA levels of inflammatory genes such as IFN-γ, TNF-α, IL-10, and IL-6 in hepatic tissues and MRC significantly attenuated this elevation. These findings suggest that ARS has detrimental effects on blood hematology and health, triggering specific inflammatory genes and indicating the genotoxicity of ARS. However, co-treatment with MYC can mitigate these negative effects. MRC exhibits a significant protective effect against ARS due to its anti-inflammatory and antioxidant properties.

Rhizospheric nano-remediation salvages arsenic genotoxicity: Zinc-oxide nanoparticles articulate better oxidative stress management, reduce arsenic uptake, and increase yield in Pisum sativum (L.)

Pea (Pisum sativum L.), a legume, has a high nutritional content, but arsenic (As) in the agro-ecosystem poses a significant bottleneck to its yield, especially in South East Asia, by severely hampering ontogeny. The present study proposes a rhizospheric nano-remediation strategy to evade As-genotoxicity and improve crop yield using biogenic zinc-oxide nanoparticles (ZnONPs). Similar to any other source of environmental stress, As-toxicity caused rapid oxidative bursts with deterioration in morpho-physiological attributes (germination rate, shoot length, and root length decreased by 62 %, 16 %, and 14.9 % respectively in the negative control, over normal control). Reactive oxygen species (ROS) accumulation (12.8 and 9-fold increase in leaves and roots) overburdened antioxidative defense, and loss of cellular homeostasis resulted in membrane damage (82.75 % increase) and electrolyte-leakage (2.6-fold increase) in negative control. The study also reveals a significant increase in nuclear area, nuclear fragmentation, and micronuclei formation in root tip cells under As-stress, indicating severe genomic instability and increased programmed cell death (3.3-fold increase in early apoptotic cells) due to leaky plasma membrane and unrepaired DNA damage. Application of ZnONPs significantly reduced As-toxicity in peas due to its adsorption in the rhizosphere, causing diminished As-uptake and better antioxidant response. Improved phytochelatin synthesis enhanced vacuolar sequestration of arsenic, which reduced As-interference. Comparatively better flowering time (7.74–19.36 % reduction in flowering delay) with greater transcript abundance of GIGANTIA (GI), CONSTANS (CO), and FLOWERING LOCUS T (FT) genes; better photosynthetic activity (1.3–1.9-fold increased chlorophyll autofluorescence); increased pollen viability; lesser genotoxicity (decreased tail DNA in comet assay) was noticed. A maximum increase of 37.5 % in pod number and seed zinc content (1.67-fold) was observed while seed arsenic content decreased under ZnONPs treatment. However, the highest dose of ZnONPs (400 mg L−1) induced NP-toxicity in pea plants under our experimental conditions, while optimum stress-alleviation was observed up to 300 mg L−1.

Protective role of eugenol on arsenic induced oxidative DNA damage and modulatory effect of GSTO2 polymorphism

Arsenic exposure is one of the major health problem worldwide. It is related with various cancers and numerous other pathological effects. The present work was aimed to study the ameliorative effects of eugenol against arsenic mediated oxidative DNA damage using Sister chromatid exchanges (SCE), comet, and lipid peroxidation assays as biomarkers. For the genotyping of GSTO2, PCR RFLP method was used. The treatment with sodium arsenite showed dose dependent increase in frequency of SCE, comet tail moment (Tm), and lipid peroxidation (TBARS levels). On administration of eugenol (10 and 20 µM) significant dose dependent decrease in the number of SCE, Tm value, and TBARS levels observed in arsenic treated cultured peripheral blood lymphocytes. Nonsignificant effects of GSTO2 polymorphism perceived on arsenic genotoxicity and eugenol antigenotoxicity. The results of the present work suggest a protective role of eugenol against arsenic induced genotoxic and oxidative damage in cultured human peripheral blood lymphocytes. Practical application Arsenic contamination of groundwater is one of the leading environmental issues worldwide. Chronic exposure of arsenic cause several health problems including tumor manifestation. The results of the present study suggest the use of indigenous dietary elements, herbs, shrubs as a safer and effective preventative medicine against heavy metal induced genotoxicity and oxidative stress.

Amplification of arsenic genotoxicity by TiO2 nanoparticles in mammalian cells: new insights from physicochemical interactions and mitochondria

Titanium dioxide nanoparticles (TiO2 NPs) have shown great adsorption capacity for arsenic (As); however, the potential impact of TiO2 NPs on the behavior and toxic responses of As remains largely unexplored. In the present study, we focused on the physicochemical interaction between TiO2 NPs and As(III) to clarify the underlying mechanisms involved in their synergistic genotoxic effect on mammalian cells. Our data showed that As(III) mainly interacted with TiO2 NPs by competitively occupying the sites of hydroxyl groups on the surface of TiO2 NP aggregates, resulting in more aggregation of TiO2 NPs. Although TiO2 NPs at concentrations used here had no cytotoxic or genotoxic effects on cells, they efficiently increased the genotoxicity of As(III) in human-hamster hybrid (AL) cells. The synergistic genotoxicity of TiO2 NPs and As(III) was partially inhibited by various endocytosis pathway inhibitors while it was completely blocked by an As(III)-specific chelator. Using a mitochondrial membrane potential fluorescence probe, a reactive oxygen species (ROS) probe together with mitochondrial DNA-depleted ρ0 AL cells, we discovered that mitochondria were essential for mediating the synergistic DNA-damaging effects of TiO2 NPs and As(III). These data provide novel mechanistic proof that TiO2 NPs enhanced the genotoxicity of As(III) via physicochemical interactions, which were mediated by mitochondria-dependent ROS.

Reduced LINE-1 methylation is associated with arsenic-induced genotoxic stress in children.

Early life exposure to arsenic has profound effect towards development of arsenic induced toxic outcomes. Some districts in the state of West Bengal, India are highly affected by arsenic, mainly through ground water. In children, not much of the toxic outcomes like dermatological lesions are observed but it is thought that the exposure leads to transient alteration in their biological processes that leads to various deleterious health effects later on. We evaluated the global methylation status by analyzing the LINE-1 methylation profile in children from arsenic exposed region between the age group 5-15years along with the cytogenetic stress induced by arsenic as measured by lymphocyte micronucleus (MN) frequency. A total of 52 arsenic exposed and 32 unexposed children were analyzed. Whole blood DNA was used to measure the LINE-1 methylation by qRT-MSP. We found a significant association of MN-frequency in exposed individuals with highly depleted LINE-1 methylation compared to the exposed individuals with near baseline (which was comparable to unexposed control) methylation index as well as with those with the hypermethylated LINE-1 promoters. From our results, we interpret that LINE-1 methylation index may serve as a potent global epigenetic mark to detect the degree of arsenic genotoxicity at a very early age. We propose that this may be utilized to determine the extent of toxic influence exerted by arsenic, from a very early age.

Role of oxidative stress in arsenic (III) induced genotoxicity in cells of meristematic tissue of Allium cepa: an in vivo study

Genotoxic effects of arsenic contaminated groundwater in Burdwan district (0 - 10μg/mL) were investigated in whole-plant roots of Allium cepa L. by use of the cellular comet assay. The results indicated that arsenic induced DNA fragmentation in a dose-dependant manner with a maximum effect at 4μg/mL and at this concentration, DNA damage time-dependently increased until 48 h. A-cellular comet assay showed that DNA breakages were not associated with a direct effect of arsenic on DNA. A. cepa that were exposed to arsenic in the presence or absence of the antioxidant NADPH-oxidase inhibitor dephenylene iodonium (DPI) showed the total inhibition of the genotoxic effect of arsenic. It revealed the major role of reactive oxygen species (ROS) in the genotoxicity of arsenic.

Natural Antioxidants Against Arsenic-Induced Genotoxicity.

Arsenic is present in water, soil, and air in organic as well as in inorganic forms. However, inorganic arsenic is more toxic than organic and can cause many diseases including cancers in humans. Its genotoxic effect is considered as one of its carcinogenic actions. Arsenic can cause DNA strand breaks, deletion mutations, micronuclei formation, DNA-protein cross-linking, sister chromatid exchange, and DNA repair inhibition. Evidences indicate that arsenic causes DNA damage by generation of reactive free radicals. Nutritional supplementation of antioxidants has been proven highly beneficial against arsenic genotoxicity in experimental animals. Recent studies suggest that antioxidants protect mainly by reducing excess free radicals via restoring the activities of cellular enzymatic as well as non-enzymatic antioxidants and decreasing the oxidation processes such as lipid peroxidation and protein oxidation. The purpose of this review is to summarize the recent literature on arsenic-induced genotoxicity and its mitigation by naturally derived antioxidants in various biological systems.

Morpho-anatomical and growth alterations induced by arsenic in Cajanus cajan (L.) DC (Fabaceae)

Arsenic (As) is a toxic element to most organisms. Studies investigating anatomic alterations due to As exposure in plants are scarce but of utmost importance to the establishment of environmental biomonitoring techniques. So, this study aimed to investigate the effects of As on the development and initial root growth in Cajanus cajan (Fabaceae), characterize and quantify the possible damages, evaluate genotoxic effects, and identify structural markers to be used in environmental bioindication. Plants were exposed hydroponically to 0.5, 1.0, 1.5, and 2.0 mg As L(-1), as sodium arsenate. Growth parameters were measured, and in the end of the exposure, root samples were analyzed for qualitative and quantitative anatomical alterations. Arsenic genotoxicity was evaluated through analysis of the mitotic index in the root apex. Compared to the control, As-treated seedlings showed an altered architecture, with significantly decreased root length (due to the lower mitotic index in the apical meristem and reduced elongation of parenchyma cells) with darkened color, and abnormal development of the root cap. A significant increase in vascular cylinder/root diameter ratio was also detected, due to the reduction of the cellular spaces in the cortex. The secondary xylem vessel elements were reduced in diameter and had sinuous walls. The severest damage was visible in the ramification zone, where uncommon division planes of phellogen and cambium cells and disintegration of the parenchyma cells adjacent to lateral roots were observed. The high sensibility of C. cajan to As was confirmed, since it caused severe damages in root growth and anatomy. The main structural markers for As toxicity were the altered root architecture, with the reduction of the elongation zone and increase of ramification zone length, and the root primordia retained within the cortex. Our results show a new approach about As toxicity and indicate that C. cajan is a promising species to be used for bioindication of environmental contamination by As.

Assessment of arsenic-induced DNA damage in goldfish by a polymerase chain reaction-based technique using random amplified polymorphic DNA markers.

Arsenic is a groundwater contaminant of global concern. It is a potent human carcinogen, and its marked genotoxic effects have been reported in several human and animal studies. The present work investigates the applicability of the random amplified polymorphic DNA (RAPD) assay to study the DNA-damaging effects of arsenic at low-level exposure in goldfish Carassius auratus. Four experimental groups of fish, A, B, C and D, were exposed to 0, 10, 50, and 1,000 µg L(-1) of arsenic, respectively, in aquaria water for 15 consecutive days. Genomic DNA extraction was followed by RAPD-polymerase chain reaction amplification for each fish separately. One arbitrary decamer primer (PUZ-19) of 33 primers used appeared as the most informative and was capable of exhibiting marked alterations in RAPD profiles between arsenic-exposed and unexposed (control) samples. Different sets of 11 loci were amplified in various experimental groups with four clear polymorphic bands by the primer PUZ-19. The X and XIII amplification loci, which were prominent in the unexposed group, failed to appear in the arsenic-exposed groups. In contrast, the I and XI RAPD bands appeared as new amplification loci in all of the exposed groups. Such alterations in genomic DNA, however, did not exhibit a clear dose-dependent tendency. The RAPD assay, because of its efficacy to unmask alterations in genomic DNA induced by arsenic at low exposure level of 10 µg L(-1), appears to be a sensitive and potential tool for detecting arsenic genotoxicity.

Arsenic is cytotoxic and genotoxic to primary human lung cells

Arsenic originates from both geochemical and numerous anthropogenic activities. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to high levels of arsenic in drinking water has been linked to bladder, lung, kidney, liver, prostate, and skin cancers. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer and few studies have considered effects in normal human lung cells. The purpose of this study was to determine the cytotoxicity and genotoxicity of arsenic in human primary bronchial fibroblast and epithelial cells. Our data show that arsenic induces a concentration-dependent decrease in cell survival after short (24h) or long (120h) exposures. Arsenic induces concentration-dependent but not time-dependent increases in chromosome damage in fibroblasts. No chromosome damage is induced after either 24h or 120h arsenic exposure in epithelial cells. Using neutral comet assay and gamma-H2A.X foci forming assay, we found that 24h or 120h exposure to arsenic induces increases in DNA double strand breaks in both cell lines. These data indicate that arsenic is cytotoxic and genotoxic to human lung primary cells but lung fibroblasts are more sensitive to arsenic than epithelial cells. Further research is needed to understand the specific mechanisms involved in arsenic-induced genotoxicity in human lung cells.

Catalase has a key role in protecting cells from the genotoxic effects of monomethylarsonous acid: A highly active metabolite of arsenic

Although it is widely known that arsenic-contaminated drinking water causes many diseases, arsenic's exact mode of action (MOA) is not fully understood. Induction of oxidative stress has been proposed as an important key event in the toxic MOA of arsenic. The authors' studies are centered on identifying a reactive species involved in the genotoxicity of arsenic using a catalase (CAT) knockout mouse model that is impaired in its ability to breakdown hydrogen peroxide (H2 O2 ). The authors assessed the induction of DNA damage using the Comet assay following exposure of mouse Cat(+/) (+) and Cat(-) (/) (-) primary splenic lymphocytes to monomethylarsonous acid (MMA(III) ) to identify the potential role of H2 O2 in mediating cellular effects of this metalloid. The results showed that the Cat(-) (/) (-) lymphocytes are more susceptible to MMA(III) than the Cat(+/) (+) lymphocytes by a small (1.5-fold) but statistically significant difference. CAT activity assays demonstrated that liver tissue has approximately three times more CAT activity than lymphocytes. Therefore, Comet assays were performed on primary Cat(+/) (+) , Cat(+/) (-) , and Cat(-) (/) (-) hepatocytes to determine if the Cat(-) (/) (-) cells were more susceptible to MMA(III) than lymphocytes. The results showed that the Cat(-) (/) (-) hepatocytes exhibit higher levels of DNA strand breakage than the Cat(+/) (+) (approximately fivefold) and Cat(+/) (-) (approximately twofold) hepatocytes exposed to MMA(III) . Electron spin resonance using 5,5-dimethyl-1-pyrroline-N-oxide as the spin-trap agent detected the generation of ·OH via MMA(III) when H2 O2 was present. These experiments suggest that CAT is involved in protecting cells against the genotoxic effects of the ·OH generated by MMA(III) .

Abstract LB-393: Genotoxicity of arsenic in primary human bronchial cells

Abstract Arsenic originates from both geochemical and numerous anthropogenic activities including mining, combustion of fossil fuels, wood preservation, agriculture and metallurgy. Exposure of the general public to significant levels of arsenic is widespread. Arsenic is a well-documented human carcinogen. Long-term exposure to low levels of arsenic in drinking water have been linked to bladder, lung, kidney, liver, prostate, and skin cancer. Among them, lung cancer is of great public concern. However, little is known about how arsenic causes lung cancer. The purpose of this study was to determine the cytotoxicity and genotoxicity in human primary bronchial fibroblasts cells (NHBF). Our data show that arsenic induces a concentration-dependent increase in cell death after acute (24 h) or chronic (120 h) exposure. 0.1, 1 and 10 uM arsenic for 24 h induced 85, 64 and 35 percent relative survival, respectively, and induced 73, 57 and 11 percent of relative survival, respectively after 120 h treatment. 24 h exposure of 5 and 10 uM arsenic induced 15 and 32 chromosome damage compared to the control. Comet assay was used to measure the formation of DNA double strand breaks. We found that 120 h exposure induced a concentration-dependent increase in double strand breaks. Future study will focus on arsenic cytotoxicity and genotoxicity in primary bronchial epithelial cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-393. doi:1538-7445.AM2012-LB-393

Genotoxic potential of arsenic at its reference dose

Arsenic, a highly hazardous contaminant in our drinking water, accounts for various toxic effects (including cancer) in human. However, intake of arsenic @0.3μgkg−1day−1 through drinking water, containing arsenic at its guideline value or maximum contaminant limit (10μgL−1), has been estimated to pose very little or no measurable risk to cancer in humans. The value also appears to be equal to the human reference dose (or index dose) of arsenic based on human skin toxicity data. The present work was a quantitative assessment of the genotoxic potential of arsenic in mice at doses equivalent to its human reference dose as well as its multiples. Significant increases in the frequencies of chromosome abnormalities in the bone marrow cells were registered over the control level upon exposure to all the doses of arsenic including its reference dose (or index dose). The assessment of arsenic genotoxicity in humans at low doses will therefore be highly instrumental in establishing a permissible limit of arsenic in drinking water.

Role of DNA polymerase beta in the genotoxicity of arsenic

Arsenic, an important hazard in the environment, is associated with human cancer and other degenerative diseases. However, the mechanisms underlying arsenic hazardous effects remain unclear. It has been reported arsenic exposure can result in increased cellular reactive oxygen species and oxidative DNA damage. This suggests DNA base excision repair (BER), the major pathway for repairing oxidative DNA damage, may be involved in combating arsenic hazardous effects. As a critical repair enzyme in BER, DNA polymerase beta (Pol β) might play an essential role in reducing arsenic toxicity. To test this hypothesis, we evaluated arsenic-induced cytotoxic and genotoxic effects under Pol β deficiency. Our results demonstrated that the viability of Pol β-deficient mouse embryonic fibroblasts was much lower than that of Pol β wild-type cells after treatment with arsenite (As(3+) ). An increased level of DNA damage and significantly delayed arsenite-induced DNA damage repair in Pol β-deficient cells indicated reduced repair of DNA lesions under Pol β deficiency. This was consistent with the increase in the frequency of micronuclei (MN), an indicator of chromosomal breakage, which was also observed in Pol β-deficient cells treated with arsenite. In contrast, cells harboring overexpressed Pol β resulted in a lower level of DNA damage and MN than Pol β wild-type cells, indicating overexpression of the enzyme can combat arsenic-induced genotoxic effects. In conclusion, our results indicate an important role for Pol β in repairing arsenite-induced DNA damage and maintaining chromosomal integrity and further suggest deficiency of BER may be involved in arsenic genotoxicity and carcinogenicity.

Biological effects of inorganic arsenic on primary cultures of rat astrocytes

It is well established that inorganic arsenic induces neurotoxic effects and neurological defects in humans and laboratory animals. The cellular and molecular mechanisms of its actions, however, remain elusive. Herein we report the effects of arsenite (NaAsO2) on primary cultures of rat astrocytes. Cells underwent induction of heat shock protein 70 only at the highest doses of inorganic arsenic (30 and 60 microM), suggesting a high threshold to respond to stress. We also investigated arsenic genotoxicity with the comet assay. Interestingly, although cells treated with 10 microM arsenite for 24 h maintained >70% viability, with respect to untreated cells, high DNA damage was already observed. Since arsenic is not known to be a direct-acting genotoxic agent, we investigated the possibility that its effects are due, in astrocytes as well, to ROS formation, as already described for other cell types. However, FACS analysis after CM-H2DCFDA staining did not evidence any significant increase of ROS production while, on the contrary, at the highest arsenite concentrations used, ROS production decreased. Concordantly, we found that, if most cells in the culture are still alive (i.e. up to 10 microM arsenite), they show a treatment-dependent increase in the concentration of SOD1. On the other hand, SOD2 concentration did not change. Finally, we found that astrocytes also synthesize PIPPin, an RNA-binding protein, the concentration of which was recently reported to change in response to stress induced by cadmium. Here we also report that, in cells exposed to high doses of arsenite, an anti-PIPPin antibody-positive faster migrating protein appears.

Arsenic: metabolism and transport mechanisms in human hepatocytes

Arsenic is a human carcinogen known to induce tumors of the urinary bladder, skin, liver and lung (Dopp et al. 2009; Beyersmann and Hartwig 2008). Genotoxicity of arsenic is not caused by direct interaction with DNA but by indirect processes, including generation of reactive oxygen species and inhibition of DNA repair processes. Drinking water represents a common source of human exposure. Tens of millions of humans in Southeast Asia routinely drink ground water with too high arsenic concentrations (Polizzotto et al. 2008). Recently, indoor combustion of high arseniccontaining coal has been reported to contribute to human exposure (Chen et al. 2007a, b, 2009; Masoudi and Saadat 2008; Lin et al. 2007). Besides its carcinogenicity, many further toxic eVects of arsenic have been reported. Examples are its compromising eVect of some immune functions (Kozul et al. 2009; Conde et al. 2007), induction of alterations in DNA methylation patterns (Jensen et al. 2009), decreased glucose tolerance (Ettinger et al. 2009), inhibition of neurite growth (Frankel et al. 2009) and oxidative myocardial and liver injury (Manna et al. 2008; Gonsebatt et al. 2007). Today, it is well accepted that the +3 methylated arsenic species are more toxic compared to the inorganic counterparts (review: Dopp et al. 2009). Methylation of inorganic arsenic mostly occurs in the liver. In this issue of the Archives of Toxicology, Miroslav Styblo from the University of North Carolina reviews metabolic pathways of arsenic in human liver particularly focusing on transporter studies performed in hepatocytes in vitro. Cultivated hepatocytes represent a well accepted in vitro system for the study of drug metabolism and toxicity (Kafert-Kasting et al. 2006; Schumann et al. 2009; Bauer et al. 2009; Godoy et al. 2009; Schug et al. 2008; Schon et al. 2008; Brulport et al. 2007; Carmo et al. 2005; Ringel et al. 2005) and have also been recommended for transporter studies (Hewitt et al. 2007). Styblo concludes that MRP2 is the most important mechanism responsible for eZux of arsenic metabolites into bile and excretion into feces. However, relatively little is known about the basolateral transport mechanisms that lead to excretion of arsenic metabolites via blood into urine.

Thresholds in Genotoxicity and Carcinogenicity: Urinary Bladder Carcinogenesis

Cancer is due to multiple alterations to DNA. Chemicals can increase the cancer risk by directly damaging DNA (DNA reactivity) or by increasing cell proliferation (DNA replications), increasing the number of opportunities for spontaneous DNA damage. Genotoxicity is a more comprehensive term than DNA reactivity. Many of the mechanisms of genotoxicity, such as clastogenicity, inhibition of DNA repair, or damage to the mitotic apparatus, produce DNA damage indirectly. These non-DNA reactive mechanisms involve interactions with proteins and mechanistically are threshold phenomena. 2-Acetylaminofluorene (AAF) is DNA reactive. Its dose response for urinary bladder DNA adduct formation is linear, whereas the tumor response is non-linear. Non-linearity is at the dose at which increased cell proliferation occurs, related to the threshold phenomenon of cytotoxicity. Non-linearity for DNA reactive carcinogens can also be produced by changes in metabolic processes of activation and/or deactivation due to saturable kinetics. Arsenic produces bladder cancer with a non-linear dose response in animal models and humans. Genotoxicity of arsenic occurs secondarily to indirect mechanisms, not DNA reactivity, it has a non-linear dose response, and the genotoxic mechanism appears to have a threshold, occurring only at doses in excess of toxic concentrations. Numerous non-genotoxic agents have been identified as bladder carcinogens in rodent models, most acting by inducing cytotoxicity with regenerative proliferation. Cytotoxicity can be produced by formation of urinary solids or by urinary reactive chemicals. Urinary solids are a defined threshold phenomenon based on the physical-chemical property of solubility. Likewise, chemical induction of cytotoxicity is a known threshold phenomenon. Non-genotoxic chemicals have a threshold dose response with respect to carcinogenesis, as do most genotoxic agents. DNA reactive chemicals have a non-linear dose response.

Genetic and epigenetic changes induced by chronic low dose exposure to arsenic of mouse testicular Leydig cells

Arsenic is an important environmental carcinogen that affects millions of people worldwide through contaminated water supplies. Genotoxicity of arsenic has been a topic of controversy. Both genetic alterations (mutations) and epigenetic changes (methylation) have been shown to play a crucial role in environmental carcinogenesis. Chronic exposure to arsenic has been shown to induce malignant transformation of mammalian cells. However, the genetic aberrations induced by arsenic in this process are unclear. The purpose of this study was to determine if both lower (1 pg/ml) and higher concentrations (100 ng/ml) of arsenic induces either mutations or methylation changes that could lead to the development of genomic instability in TM3 cells, immortalized Leydig cells derived from normal mouse testis. Two independent exposure times were used in this study which resulted in cells of 33 and 100 generations in age. Arsenic-induced genetic and epigenetic changes were screened at a genome-wide level by random amplified polymorphic DNA (RAPD), also known as AP-PCR method with undigested DNA as well as DNA digested by the methylation sensitive isosizomeric restriction enzymes MSPI and HpaII and untreated controls. Changes in the DNA fingerprint of both, the restriction enzyme digested DNA (indicating methylation changes) as well as undigested DNA (indicating mutations) from arsenic-treated (low as well as high dose) samples were observed as compared to their controls. Thus, this study provides the first evidence at DNA sequence level for mutagenic potential of arsenic. Further characterization of these altered genomic regions is underway. The understanding of these genetic and epigenetic changes in arsenic-induced carcinogenesis will provide a basis for better interventional approaches in both the treatment and prevention of arsenic-induced cancer.

Arsenic-Induced Genotoxic and Cytotoxic Effects in Human Keratinocytes, Melanocytes and Dendritic Cells

Arsenical keratosis and skin cancer are among the most common health effects associated with acute and chronic exposures to arsenic. This study examines the acute and chronic dose-responses of arsenic in established human cell lines using keratinocytes (HaCaT), melanocytes (CRL1675) and dendritic cells (THP-1 + A23187). Chronic conditions were established by treating the three cell lines with at least 8 passages in 0.2 microg/mL arsenic trioxide. Cytotoxicity was assessed using the fluorescein diacetate assay after 72 hrs of exposure. Single cell gel electrophoresis (Comet assay) was used to measure DNA damage. Acute exposure to arsenic had LD10 and LD25 values of 0.38 microg/mL and 3.0 microg/mL for keratinocytes; 0.19 microg/mL and 0.38 microg/mL for melanocytes; and 0.38 microg/mL and 0.75 microg/mL for dendritic cells. Cytotoxicity assays for chronically exposed cells resulted in LD10, and LD25 values of 0.4 microg/mL and 0.8 microg/mL for keratinocytes; 0.10 microg/mL and 0.20 microg/mL for melanocytes; and 0.10 microg/mL and 1.0 microg/mL for dendritic cells. The Comet assay showed that arsenic was highly genotoxic to the three cell lines. No significant differences (p > 0.05) in DNA cleavage were observed between acute and chronic exposures. In acute exposure arsenic genotoxicity was more severe with dendritic cells while melanocytes were more sensitive to arsenic cytotoxicity. Similarly, chronically exposed dendritic cells showed the maximum genotoxic damage while melanocytes were more sensitive to arsenic cytotoxicity. In conclusion, this research shows that arsenic is dermatotoxic, showing a high degree of genotoxicity and cytotoxicity to skin cells.

Dietary folate deficiency enhances induction of micronuclei by arsenic in mice.

Folate deficiency increases background levels of DNA damage and can enhance the genotoxicity of chemical agents. Arsenic, a known human carcinogen present in drinking water supplies around the world, induces chromosomal and DNA damage. The effect of dietary folate deficiency on arsenic genotoxicity was evaluated using a mouse peripheral blood micronucleus (MN) assay. In duplicate experiments, male C57Bl/6J mice were fed folate-deficient or folate-sufficient diets for 7 weeks. During week 7, mice on each diet were given four consecutive daily doses of sodium arsenite (0, 2.5, 5, or 10 mg/kg) via oral gavage. Over the course of the study the folate-deficient diet produced an approximate 60% depletion of red blood cell folate. Folate deficiency by itself was associated with small but significant increases in MN in normochromatic erythrocytes (NCEs) and polychromatic erythrocytes (PCEs). Arsenic exposure was associated with significant increases in MN-PCEs in both folate-deficient and folate-sufficient mice. MN-PCE frequencies at the 10 mg/kg dose of arsenic were increased 4.5-fold over vehicle control in folate-deficient mice and 2.1-fold over control in folate-sufficient mice. At the 5 and 10 mg/kg doses of arsenic, MN-PCE levels were significantly higher (1.3-fold and 2.4-fold, respectively) in folate-deficient mice compared to folate-sufficient mice. Very few MN from either control or treated animals in either experiment exhibited kinetochore immunostaining, suggesting that the MN were derived from chromosome breakage rather than from whole chromosome loss. These results indicate that folate deficiency enhances arsenic-induced clastogenesis at doses of 5 mg/kg and higher.