Assessing testicular germ cell DNA damage in the comet assay; introduction of a proof-of-concept.

The in vivo comet assay is widely used to measure genotoxicity; however, the current OECD test guideline (TG 489) does not recommend using the assay to assess testicular germ cells, due to the presence of testicular somatic cells. An adapted approach to specifically assess testicular germ cells within the comet assay is certainly warranted, considering regulatory needs for germ cell-specific genotoxicity data in relation to the increasing global production of and exposure to potentially hazardous chemicals. Here, we provide a proof-of-concept to selectively analyze round spermatids and primary spermatocytes, distinguishing them from other cells of the testicle. Utilizing the comet assay recordings of DNA content (total fluorescence intensity) and DNA damage (% tail intensity) of individual comets, we developed a framework to distinguish testicular cell populations based on differences in DNA content/ploidy and appearance. Haploid round spermatid comets are identified through (1) visual inspection of DNA content distributions, (2) setting DNA content thresholds, and (3) modeling DNA content distributions using a normal mixture distribution function. We also describe an approach to distinguish primary spermatocytes during comet scoring, based on their high DNA content and large physical size. Our concept allows both somatic and germ cells to be analyzed in the same animal, adding a versatile, sensitive, rapid, and resource-efficient assay to the limited genotoxicity assessment toolbox for germ cells. An adaptation of TG 489 facilitates accumulation of valuable information regarding distribution of substances to germ cells and their potential for inducing germ cell gene mutations and structural chromosomal aberrations.

Chemical carcinogen safety testing: OECD expert group international consensus on the development of an integrated approach for the testing and assessment of chemical non-genotoxic carcinogens.

While regulatory requirements for carcinogenicity testing of chemicals vary according to product sector and regulatory jurisdiction, the standard approach starts with a battery of genotoxicity tests (which include mutagenicity assays). If any of the in vivo genotoxicity tests are positive, a lifetime rodent cancer bioassay may be requested, but under most chemical regulations (except plant protection, biocides, pharmaceuticals), this is rare. The decision to conduct further testing based on genotoxicity test outcomes creates a regulatory gap for the identification of non-genotoxic carcinogens (NGTxC). With the objective of addressing this gap, in 2016, the Organization of Economic Cooperation and Development (OECD) established an expert group to develop an integrated approach to the testing and assessment (IATA) of NGTxC. Through that work, a definition of NGTxC in a regulatory context was agreed. Using the adverse outcome pathway (AOP) concept, various cancer models were developed, and overarching mechanisms and modes of action were identified. After further refining and structuring with respect to the common hallmarks of cancer and knowing that NGTxC act through a large variety of specific mechanisms, with cell proliferation commonly being a unifying element, it became evident that a panel of tests covering multiple biological traits will be needed to populate the IATA. Consequently, in addition to literature and database investigation, the OECD opened a call for relevant assays in 2018 to receive suggestions. Here, we report on the definition of NGTxC, on the development of the overarching NGTxC IATA, and on the development of ranking parameters to evaluate the assays. Ultimately the intent is to select the best scoring assays for integration in an NGTxC IATA to better identify carcinogens and reduce public health hazards.

In vivo Comet assay--statistical analysis and power calculations of mice testicular cells.

The in vivo Comet assay is a sensitive method for evaluating DNA damage. A recurrent concern is how to analyze the data appropriately and efficiently. A popular approach is to summarize the raw data into a summary statistic prior to the statistical analysis. However, consensus on which summary statistic to use has yet to be reached. Another important consideration concerns the assessment of proper sample sizes in the design of Comet assay studies. This study aims to identify a statistic suitably summarizing the % tail DNA of mice testicular samples in Comet assay studies. A second aim is to provide curves for this statistic outlining the number of animals and gels to use. The current study was based on 11 compounds administered via oral gavage in three doses to male mice: CAS no. 110-26-9, CAS no. 512-56-1, CAS no. 111873-33-7, CAS no. 79-94-7, CAS no. 115-96-8, CAS no. 598-55-0, CAS no. 636-97-5, CAS no. 85-28-9, CAS no. 13674-87-8, CAS no. 43100-38-5 and CAS no. 60965-26-6. Testicular cells were examined using the alkaline version of the Comet assay and the DNA damage was quantified as % tail DNA using a fully automatic scoring system. From the raw data 23 summary statistics were examined. A linear mixed-effects model was fitted to the summarized data and the estimated variance components were used to generate power curves as a function of sample size. The statistic that most appropriately summarized the within-sample distributions was the median of the log-transformed data, as it most consistently conformed to the assumptions of the statistical model. Power curves for 1.5-, 2-, and 2.5-fold changes of the highest dose group compared to the control group when 50 and 100 cells were scored per gel are provided to aid in the design of future Comet assay studies on testicular cells.

The influence of the number of cells scored on the sensitivity in the comet assay.

The impact on the sensitivity of the in vitro comet assay by increasing the number of cells scored has only been addressed in a few studies. The present study investigated whether the sensitivity of the assay could be improved by scoring more than 100 cells. Two cell lines and three different chemicals were used: Caco-2 cells were exposed to ethylmethane sulfonate and hydrogen peroxide in three concentrations, and HepG2 cells were exposed to ethylmethane sulfonate, hydrogen peroxide and benzo[a]pyrene in up to four concentrations, in four to five independent experiments. The scoring was carried out by means of a fully automated scoring system and the results were analyzed by evaluating the % tail DNA of 100-700 randomly selected cells for each slide consisting of two gels. By increasing the number of cells scored, the coefficients of variance decreased, leading to an improved sensitivity of the assay. A two-way ANOVA analysis of variance showed that the contribution from the two variables "the number of cells scored" and "concentration" on the total variation in the coefficients of variance dataset was statistically significant (p<0.05). The increase in sensitivity was demonstrated by the possibility to detect an increase in % tail DNA with statistical significance at lower concentrations. The results indicated that for low levels of DNA damage, below 9% tail DNA, scoring of 600 cells increased the sensitivity compared with scoring of 100 cells. For relatively low levels of DNA damage, about 9-16% tail DNA, scoring of 300 cells increased the sensitivity. Thus, the recommendation for the optimum number of cells scored would be 600 and 300 for low and relatively low levels of DNA damage, respectively. The findings from this study could be particularly important for bio-monitoring studies where small differences in DNA-damage levels could be relevant.

Oxidative stress, DNA damage, and inflammation induced by ambient air and wood smoke particulate matter in human A549 and THP-1 cell lines.

Combustion of biomass and wood for residential heating and/or cooking contributes substantially to both ambient air and indoor levels of particulate matter (PM). Toxicological characterization of ambient air PM, especially related to traffic, is well advanced, whereas the toxicology of wood smoke PM (WSPM) is poorly assessed. We assessed a wide spectrum of toxicity end points in human A549 lung epithelial and THP-1 monocytic cell lines comparing WSPM from high or low oxygen combustion and ambient PM collected in a village with many operating wood stoves and from a rural background area. In both cell types, all extensively characterized PM samples (1.25-100 µg/mL) induced dose-dependent formation of reactive oxygen species and DNA damage in terms of strand breaks and formamidopyrimidine DNA glycosylase sites assessed by the comet assay with WSPM being most potent. The WSPM contained more polycyclic aromatic hydrocarbons (PAH), less soluble metals, and expectedly also had a smaller particle size than PM collected from ambient air. All four types of PM combined increased the levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine dose-dependently in A549 cells, whereas there was no change in the levels of etheno-adducts or bulky DNA adducts. Furthermore, mRNA expression of the proinflammatory genes monocyte chemoattractant protein-1, interleukin-8, and tumor necrosis factor-α as well as the oxidative stress gene heme oxygenase-1 was upregulated in the THP-1 cells especially by WSPM and ambient PM sampled from the wood stove area. Expression of oxoguanine glycosylase 1, lymphocyte function-associated antigen-1, and interleukin-6 did not change. We conclude that WSPM has small particle size, high level of PAH, low level of water-soluble metals, and produces high levels of free radicals, DNA damage as well as inflammatory and oxidative stress response gene expression in cultured human cells.

Genotoxicity of unmodified and organo-modified montmorillonite.

The natural clay mineral montmorillonite (Cloisite) Na+) and an organo-modified montmorillonite (Cloisite 30B) were investigated for genotoxic potential as crude suspensions and as suspensions filtrated through a 0.2-microm pore-size filter to remove particles above the nanometre range. Filtered and unfiltered water suspensions of both clays did not induce mutations in the Salmonella/microsome assay at concentrations up to 141microg/ml of the crude clay, using the tester strains TA98 and TA100. Filtered and unfiltered Cloisite) Na+ suspensions in culture medium did not induce DNA strand-breaks in Caco-2 cells after 24h of exposure, as tested in the alkaline comet assay. However, both the filtered and the unfiltered samples of Cloisite 30B induced DNA strand-breaks in a concentration-dependent manner and the two highest test concentrations produced statistically significantly different results from those seen with control samples (p<0.01 and p<0.001) and (p<0.05 and p<0.01), respectively. The unfiltered samples were tested up to concentrations of 170microg/ml and the filtered samples up to 216microg/ml before filtration. When tested in the same concentration range as used in the comet assay, none of the clays produced ROS in a cell-free test system (the DCFH-DA assay). Inductively coupled plasma mass-spectrometry (ICP-MS) was used to detect clay particles in the filtered samples using aluminium as a tracer element characteristic to clay. The results indicated that clay particles were absent in the filtered samples, which was independently confirmed by dynamic light-scattering measurements. Detection and identification of free quaternary ammonium modifier in the filtered sample was carried out by HPLC-Q-TOF/MS and revealed a total concentration of a mixture of quaternary ammonium analogues of 1.57microg/ml. These findings suggest that the genotoxicity of organo-modified montmorillonite was caused by the organo-modifier. The detected organo-modifier mixture was synthesized and comet-assay results showed that the genotoxic potency of this synthesized organo-modifier was in the same order of magnitude at equimolar concentrations of organo-modifier in filtrated Cloisite) 30B suspensions, and could therefore at least partly explain the genotoxic effect of Cloisite) 30B.

Inflammation but no DNA (deoxyribonucleic acid) damage in mice exposed to airborne dust from a biofuel plant.

OBJECTIVES: Particles in ambient air are associated with such health effects as lung diseases and cancer of the lung. Exposure to bioaerosols has been found to be associated with respiratory symptoms. The toxic properties of exposure to combustion and bioaerosol particles from biofuel plants have not been studied in detail. This study investigated whether exposure to dust from biofuel plants induces DNA (deoxyribonucleic acid) damage and inflammation in exposed mice. METHODS: DNA damage and inflammation were evaluated in mice exposed through the intratracheal installation of airborne dust collected at a biofuel plant at the straw storage hall and in the boiler room. The mice were given either a single dose of dust (18 or 54 microg) or four doses of 54 microg on each of four consecutive days. Control mice were exposed to a 0.9% sodium chloride solution. RESULTS: In the mice exposed to 4 x 54 microg of dust, the lung tissue mRNA (messenger ribonucleic acid) levels of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-2 (MIP-2) were increased more than 10-fold if the dust was from the boiler room and 30- to 60-fold if the dust came from the straw storage hall. The levels of DNA strand breaks in broncheoalveolar lavage (BAL) cells from the mice exposed to dust did not differ from those in the control samples. CONCLUSIONS: The results indicate that the instillation of dust from a biofuel plant, at doses corresponding to 2 weeks of exposure to human endotoxins, results in a strong inflammatory response without detectable DNA damage in BAL cells. The dust from the straw storage hall induced the strongest inflammatory response and had the highest concentration of most microbial components.

Genotoxicity, inflammation and physico-chemical properties of fine particle samples from an incineration energy plant and urban air.

Airborne particulate matter (PM) was sampled by use of an electrostatic sampler in an oven hall and a receiving hall in a waste-incineration energy plant, and from urban air in a heavy-traffic street and from background air in Copenhagen. PM was sampled for 1-2 weeks, four samples at each site. The samples were extracted and examined for mutagenicity in Salmonella typhimurium strains TA98, YG1041 and YG5161, for content of inorganic elements and for the presence of eight polycyclic aromatic hydrocarbons. The induction of IL-6 and IL-8 mRNA expression and the presence of DNA damage - tested by the comet assay - were determined after 24-h incubations with human A549 lung epithelial cells. The PM(2.5) concentration was about twofold greater in the oven hall than in the receiving hall. The particle size distribution in the receiving hall was similar to that in street air (maximum mode at about 25nm), but the distribution was completely different in the oven hall (maximum mode at about 150nm). Also chemically, the samples from the oven hall were highly different from the other samples. PM extracts from the receiving hall, street and background air were more mutagenic than the PM extracts from the oven hall. PM from all four sites caused similar levels of DNA damage in A549 cells; only the oven hall samples gave results that were statistically significantly different from those obtained with street-air samples. The receiving hall and the urban air samples were similarly inflammatory (relative IL-8 mRNA expression), whereas the oven hall did not cause a statistically significant increase in IL-8 mRNA expression. A principal component analysis separated the oven hall and the receiving hall by the first principal component. These two sites were separated from street and background air with the second principal component. Several clusters of constituents were identified. One cluster consisted of all the polycyclic aromatic hydrocarbons (PAH), several groups of metals and one group of the biological endpoints (DNA damage, IL-6 and IL-8 mRNA expression). The PAH and the inorganic content of the air in the receiving hall may be due to vehicle emissions and suspended waste particles. The inorganic content in the street and background air may have been influenced by break wear, road emissions and long-range transport. The results from a partial least-square regression analysis predicted that both PAHs and a group of metals including Fe and Mn contributed to IL-6 and IL-8 induction. Only Mn and Sr were predicted to influence DNA damage statistically significantly. Among the PAHs only chrysene had influence on DNA damage. The PM from the oven hall was markedly different from the PM at other locations in particle size distribution, chemical composition and the resulting biological effects when A549 cells were incubated with the PM. These characteristics and observations in the oven hall indicated that the PM source was oven exhaust, which was well combusted.