Atthe International Workshop on Genotoxicity Test Procedures (IWGTP) held in Washington, DC, March 25-26, 1999, an expert panel met to develop guidelines for the use of the single-cell gel (SCG)/Comet assay in genetic toxicology. The expert panel reached a consensus that the optimal version of the Comet assay for identifying agents with genotoxic activity was the alkaline (pH > 13) version of the assay developed by Singh et al. [1988]. The pH > 13 version is capable of detecting DNA single-strand breaks (SSB), alkali-labile sites (ALS), DNA-DNA/DNA-protein cross-linking, and SSB associated with incomplete excision repair sites. Relative to other genotoxicity tests, the advantages of the SCG assay include its demonstrated sensitivity for detecting low levels of DNA damage, the requirement for small numbers of cells per sample, its flexibility, its low costs, its ease of application, and the short time needed to complete a study. The expert panel decided that no single version of the alkaline (pH > 13) Comet assay was clearly superior. However, critical technical steps within the assay were discussed and guidelines developed for preparing slides with agarose gels, lysing cells to liberate DNA, exposing the liberated DNA to alkali to produce single-stranded DNA and to express ALS as SSB, electrophoresing the DNA using pH > 13 alkaline conditions, alkali neutralization, DNA staining, comet visualization, and data collection. Based on the current state of knowledge, the expert panel developed guidelines for conducting in vitro or in vivo Comet assays. The goal of the expert panel was to identify minimal standards for obtaining reproducible and reliable Comet data deemed suitable for regulatory submission. The expert panel used the current Organization for Economic Co-operation and Development (OECD) guidelines for in vitro and in vivo genetic toxicological studies as guides during the development of the corresponding in vitro and in vivo SCG assay guidelines. Guideline topics considered included initial considerations, principles of the test method, description of the test method, procedure, results, data analysis and reporting. Special consideration was given by the expert panel to the potential adverse effect of DNA degradation associated with cytotoxicity on the interpretation of Comet assay results. The expert panel also discussed related SCG methodologies that might be useful in the interpretation of positive Comet data. The related methodologies discussed included: (1) the use of different pH conditions during electrophoreses to discriminate between DNA strand breaks and ALS; (2) the use of repair enzymes or antibodies to detect specific classes of DNA damage; (3) the use of a neutral diffusion assay to identify apoptotic/necrotic cells; and (4) the use of the acellular SCG assay to evaluate the ability of a test substance to interact directly with DNA. The alkaline (pH > 13) Comet assay guidelines developed by the expert panel represent a work in progress. Additional information is needed before the assay can be critically evaluated for its utility in genetic toxicology. The information needed includes comprehensive data on the different sources of variability (e.g., cell to cell, gel to gel, run to run, culture to culture, animal to animal, experiment to experiment) intrinsic to the alkaline (pH > 3) SCG assay, the generation of a large database based on in vitro and in vivo testing using these guidelines, and the results of appropriately designed multilaboratory international validation studies.

https://onlinelibrary.wiley.com/doi/epdf/10.1002/%28SICI%291098-2280%282000%2935%3A3%3C206%3A%3AAID-EM8%3E3.0.CO%3B2-J

Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing.

Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries: a review

Arsenic toxicity and potential mechanisms of action

https://stacks.cdc.gov/view/cdc/37676/cdc_37676_DS1.pdf

Toxicological Profile for Lead

Arsenic (As) is a toxic metalloid element that is present in air, water and soil. Inorganic arsenic tends to be more toxic than organic arsenic. Examples of methylated organic arsenicals include monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)]. Reactive oxygen species (ROS)-mediated oxidative damage is a common denominator in arsenic pathogenesis. In addition, arsenic induces morphological changes in the integrity of mitochondria. Cascade mechanisms of free radical formation derived from the superoxide radical, combined with glutathione-depleting agents, increase the sensitivity of cells to arsenic toxicity. When both humans and animals are exposed to arsenic, they experience an increased formation of ROS/RNS, including peroxyl radicals (ROO•), the superoxide radical, singlet oxygen, hydroxyl radical (OH•) via the Fenton reaction, hydrogen peroxide, the dimethylarsenic radical, the dimethylarsenic peroxyl radical and/or oxidant-induced DNA damage. Arsenic induces the formation of oxidized lipids which in turn generate several bioactive molecules (ROS, peroxides and isoprostanes), of which aldehydes [malondialdehyde (MDA) and 4-hydroxy-nonenal (HNE)] are the major end products. This review discusses aspects of chronic and acute exposures of arsenic in the etiology of cancer, cardiovascular disease (hypertension and atherosclerosis), neurological disorders, gastrointestinal disturbances, liver disease and renal disease, reproductive health effects, dermal changes and other health disorders. The role of antioxidant defence systems against arsenic toxicity is also discussed. Consideration is given to the role of vitamin C (ascorbic acid), vitamin E (α-tocopherol), curcumin, glutathione and antioxidant enzymes such as superoxide dismutase, catalase and glutathione peroxidase in their protective roles against arsenic-induced oxidative stress.

/pdf/arsenic-toxicity-oxidative-stress-and-human-disease-2lp5eu82d7.pdf

Arsenic: toxicity, oxidative stress and human disease.

The concentrations of four chemical species of arsenic in urine were observed with time, after ingestion of three different chemical species of arsenic. The arsenic-rich substances ingested, including arsenite-rich wine, arsenate-rich drinking water, and crab meat which contained organo-arsenic compounds. After ingestion of arsenite-rich wine, approximately 10% of the arsenic was excreted as arsenite, but the majority of the arsenic was methylated to methylarsonic acid and dimethylarsinic acid and excreted. After ingestion of arsenate-rich water, elevated levels of both arsenate and dimethylarsinic acid were observed. When crab meat was ingested, none of these four arsenic species were observed at elevated levels until the urine was heated in 2N NaOH. After the hot base digestion, high levels of dimethylarsinic acid were detected in these samples. The apparent biological half-lives were on the order of 10 hr for inorganic arsenic and 30 hr for the methylated arsenic forms.

/pdf/changes-in-the-chemical-speciation-of-arsenic-following-153ycr333p.pdf

Changes in the chemical speciation of arsenic following ingestion by man.

It has been proposed that acid volatile sulfide (AVS) is an important sediment phase for determining the toxicity of certain trace metals. By evaluating the ratio of the molar quantities of simultaneously extracted metal (SEM) to AVS, the toxicity of metals to organisms in contact with sediment can be predicted. This study examines the role of AVS in predicting the toxicity of zinc, lead, and copper in marine sediments.

Sediment samples were titrated with zinc, lead, and copper and subsequently analyzed for SEM, pore-water (PW) metal, and AVS retention. In most cases, metal was not detected in the pore waters until the AVS was exceeded, suggesting that AVS is an adequate measure of the metal-binding capacity of a sediment. The [SEM]-to-[AVS] ratios were calculated and toxicities predicted for each spiking concentration where [SEM]/[AVS] > 1. A 10-d, flow-through, acute bioassay using the marine poly-chaete Capitella capitata was conducted to examine the prediction of toxicity from the metal titrations and the bioassay sediment chemistry data. In most cases, mortalities occurred as predicted. AVS and the [SEM]-to-[AVS] ratio proved useful as predictors of toxicity for zinc, lead, and perhaps copper. Another tool for predicting metal toxicity in sediments may be the [PW]/LC50 value; in every case where this ratio was >1, mortalities occurred.

Relationship between acid volatile sulfide and the toxicity of zinc, lead and copper in marine sediments

This study evaluated the effect of hepatic methyl donor status on the ability of sodium arsenite (25, 50 and 100 mg/kg) administered by gavage once or on four consecutive days to induce DNA damage in male B6C3F1 mice Maintenance on a choline-deficient (CD) diet prior to treatment resulted in mice with hepatic methyl donor deficiency (HMDD) and altered arsenical metabolism, as demonstrated by a decreased total urinary excretion of inorganic and organic arsenicals The alkaline (pH > 13) Single Cell Gel (SCG) assay was used to evaluate for the induction of DNA damage (single strand breaks, alkali labile sites, DNA crosslinking) in blood leukocytes, liver parenchymal cells, and cells sampled from bladder, lung, and skin, while the bone marrow erythrocyte micronucleus (MN) assay was used to assess for the induction of chromosomal damage in bone marrow cells Treatment with sodium arsenite once or four times induced a significant decrease in DNA migration (indicative of DNA crosslinking) in bladder and liver parenchymal cells of hepatic methyl donor sufficient (HMDS) mice, but in skin cells of HMDD mice Both HMDD and HMDS mice exhibited a significant increase in the frequency of micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow following four, but not following one, treatments However, the positive response occurred at a lower dose for HMDS mice and, in these mice, bone marrow toxicity, as demonstrated by a significant reduction in the percentage of PCE, was present also These results indicate that hepatic methyl donors deficiency significantly decreases the total urinary excretion of orally administered sodium arsenite and markedly modulates target organ arsenic-induced DNA damage, with an apparent shift from liver and bladder to skin

Effect of hepatic methyl donor status on urinary excretion and DNA damage in B6C3F1 mice treated with sodium arsenite.

The sea-surface microlayer is an important interface between the atmosphere and ocean and a collection point for many anthropogenic materials including potentially toxic metals. We developed a glass plate sampler to collect the upper 30 to 55 micrometers of the sea surface. Samples of the microlayer and subsurface bulk water from an urban and rural bay were analysed for concentrations of Pb, Zn, Cu, Cd and Fe. Metal concentrations in both the microlayer and bulk water were generally 2 to 15 times greater in the urban than in the rural bay. Concentrations of metals in the microlayer of both bays averaged 6 to 65 times greater than those in the bulk water. In the urban bay, microlayer concentrations of Pb, Zn and Cu from 10 to > 100 μg 1−1 were common. Measured microlayer metals concentrations agree well with those predicted from atmospheric deposition rates using a previously derived empirical model developed from laboratory microcosm studies. Further work will be required to determine whether or not these high microlayer metal concentrations contain significant biologically available fractions which could impact fisheries recruitment of larval icthyoneuston.

E.A. Crecelius

Papers

Changes in the chemical speciation of arsenic following ingestion by man.

Relationship between acid volatile sulfide and the toxicity of zinc, lead and copper in marine sediments

Effect of hepatic methyl donor status on urinary excretion and DNA damage in B6C3F1 mice treated with sodium arsenite.

Sea-surface microlayer metals enrichments in an urban and rural bay☆

Aquatic surface microlayer contamination in chesapeake bay