Hexavalent chromium [Cr(VI)] is a well-established human carcinogen, yet the mechanisms by which it leads to carcinogenic outcomes is still unclear. As a driving factor in its carcinogenic mechanism, Cr(VI) causes DNA double strand breaks and break-repair deficiency, leading to the development of chromosome instability. Therefore, the aim of this review is to discuss studies assessing Cr(VI)-induced DNA double strand breaks, chromosome damage and instability, and neoplastic transformation including cell culture, experimental animal, human pathology and epidemiology studies. Recent findings confirm Cr(VI) induces DNA double strand breaks, chromosome instability and neoplastic transformation in exposed cells, animals and humans, emphasizing these outcomes as key steps in the mechanism of Cr(VI) carcinogenesis. Moreover, recent findings suggest chromosome instability is a key phenotype in Cr(VI)-neoplastically transformed clones and is an inheritable and persistent phenotype in exposed cells, once more suggesting chromosome instability as central in the carcinogenic mechanism. Although limited, some studies have demonstrated DNA damage and epigenetic modulation are also key outcomes in biopsies from chromate workers that developed lung cancer. Additionally, we also summarized new studies showing Cr(VI) causes genotoxic and clastogenic effects in cells from wildlife, such as sea turtles, whales, and alligators. Overall, across the literature, it is clear that Cr(VI) causes neoplastic transformation and lung cancer. Many studies measured Cr(VI)-induced increases in DNA double strand breaks, the most lethal type of breaks clearly showing that Cr(VI) is genotoxic. Unrepaired or inaccurately repaired breaks lead to the development of chromosome instability, which is a common phenotype in Cr(VI) exposed cells, animals, and humans. Indeed, many studies show Cr(VI) induces both structural and numerical chromosome instability. Overall, the large body of literature strongly supports the conclusion that Cr(VI) causes DNA double strand breaks, inhibits DNA repair and chromosome instability, which are key to the development of Cr(VI)-induced cell transformation.

Ranked as a "top 10 cause of death", chronic liver disease accounts for millions of deaths annually. The prevalence of the most prominent liver disease, metabolic dysfunction-associated steatotic liver disease (MASLD), has doubled over the past 20 years and continues to rise. Growing in parallel are environmental chemical exposures, emergingas key risk factors for liver disease. Hexavalent chromium [Cr(VI)] is the #1 chemical hazard in U.S. drinking water and is associated with increased liver disease incidence, including liver cancer. How Cr(VI) contributes to liver disease is poorly understood and no studies have considered whether Cr(VI) exposure together with high-fat diet, a well-established MASLD risk factor, drives liver disease. Thus, we developed an in vivo model to study the impact of Cr(VI) and high-fat diet on MASLD. Male Sprague Dawley rats were exposed to Cr(VI) in drinking water (0, 0.05, or 0.1 mg/L - levels that reflect the World Health Organization and U.S. Environmental Protection Agency maximum contaminant levels, respectively) and fed a normal or high-fat diet for 90 days. We observed high-fat diet significantly increased body mass, plasma lipoproteins and cholesterol, liver injury and hepatic triglycerides in rats and these outcomes were exacerbated by exposure to Cr(VI). Cr(VI) alone increased hepatic expression of alpha fetoprotein, a liver cancer biomarker, with evidence suggesting Cr(VI) and high-fat diet together increased these levels more than either exposure alone. Our results indicate Cr(VI) at very low levels combined with high-fat diet worsens liver disease, emphasizing the need to reconsider current Cr(VI) drinking water regulations.

BACKGROUND:Hexavalent chromium [Cr(VI)] is a known human lung carcinogen with widespread exposure risks, yet how it causes cancer is still unclear. Cr(VI) causes dyshomeostasis of essential metals in the brains of exposed rats and is considered a biomarker of lung cancer, yet whether Cr(VI) causes metal dyshomeostasis in the lung is unknown. Cr(VI) particles impact at lung bifurcations causing tumors at these sites, indicating the bronchial branching is a key consideration for its toxicity. This study chose guinea pigs because, like humans, they have a dichotomous bronchiole branching pattern. The aim of this study was to investigate Cr accumulation and essential metal dyshomeotasis in the lung, liver and blood. METHODS:We acutely and subchronically exposed guinea pigs to zinc chromate through oropharyngeal aspiration. We collected lung, liver and blood, and quantified metal levels using inductively coupled plasma-mass spectrometry. RESULTS:Our data indicate oropharyngeal Cr(VI) exposure results in Cr accumulation in the lung and liver. We also measured zinc levels, and despite using zinc chromate as our representative Cr(VI) compound we observed no changes. This study is the first study to consider Cr(VI)-induced essential metal dyshomeostasis in the lung, liver and blood, and we found Cr(VI) exposure changed the levels of essential metals like potassium, sodium, calcium, magnesium, iron, copper, cobalt, manganese, selenium and molybdenum. CONCLUSIONS:This study established a new relevant model to study Cr(VI)-carcinogenesis and our results suggest metal dyshomeostasis could be part of the Cr(VI) carcinogenic mechanism and these effects should be considered in future mechanistic studies.

Lung cancer is an important human health concern because of its high mortality rate, with many cases caused by environmental chemicals other than tobacco. Particulate hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen, but how Cr(VI) induces lung cancer is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driving factor in Cr(VI)-induced lung cancer. Our previous studies in cultured human lung cells showed that particulate Cr(VI) induces DNA double-strand breaks during the late S and G2 phases of the cell cycle, which are repaired by homologous recombination, one of the main repair pathways of DNA double-strand breaks. Our previous data showed that prolonged exposure to Cr(VI) inhibits homologous recombination repair by targeting RAD51, a key protein that mediates homologous recombination. Therefore, particulate Cr(VI)-induced DNA damage combined with failure of DNA repair can lead to chromosome instability. In this study we translated these results to rat lung tissue and lung tumor tissue from Cr(VI)-exposed workers. Wistar rats were exposed to zinc chromate in a saline solution or saline alone by oropharyngeal aspiration with a single dose repeated weekly for 90 days. We observed DNA double-strand breaks increased in a concentration-dependent manner, but homologous recombination repair decreased in rat lungs after 90 days of exposure. Notably, these effects were more pronounced in bronchioles than alveoli. We also considered these effects in Cr(VI)-associated human lung tumors and observed increased DNA double-strand breaks and reduced RAD51 levels in lung tumor tissue compared with adjacent normal lung tissue. Thus, Cr(VI)-induced induction of DNA double-strand breaks, and inhibition of homologous recombination repair translates from cultured cells to experimental animals, normal lung tissue adjacent to the tumor, and Cr(VI)-associated human lung tumors.

Hexavalent chromium [Cr(VI)] is a known lung carcinogen and a driving mechanism in human lung cells for Cr(VI)-induced lung cancer is chromosome instability, caused by prolonged Cr(VI) exposure inducing DNA double-strand breaks, while simultaneously inhibiting the repair of these breaks. In North Atlantic right whales, Cr(VI) induces breaks but does not inhibit repair. It is unclear if this repair inhibition is specific to human lung cells or occurs in other species, as it has only been considered in humans and North Atlantic right whales. We evaluated these outcomes in rodent cells, as rodents are an experimental model for metal-induced lung carcinogenesis. We used a guinea pig lung fibroblast cell line, JH4 Clone 1, and rat lung fibroblasts. Cells were exposed to two different particulate Cr(VI) compounds, ranging from 0 to 0.5 ug/cm2, for 24 or 120 h and assessed for cytotoxicity, DNA double-strand breaks, and DNA double-strand break repair. Both particulate Cr(VI) compounds induced a concentration-dependent increase in cytotoxicity and DNA double-strand breaks after acute and prolonged exposures. Notably, while the repair of Cr(VI)-induced DNA double-strand breaks increased after acute exposure, the repair of these breaks was inhibited after prolonged exposure. These results are consistent with outcomes in human lung cells indicating rodent cells respond like human cells, while whale cells have a markedly different response.

Chromosome instability, a hallmark of lung cancer, is a driving mechanism for hexavalent chromium [Cr(VI)] carcinogenesis in humans. Cr(VI) induces structural and numerical chromosome instability in human lung cells by inducing DNA double-strand breaks and inhibiting homologous recombination repair and causing spindle assembly checkpoint (SAC) bypass and centrosome amplification. Great whales are long-lived species with long-term exposures to Cr(VI) and accumulate Cr in their tissue, but exhibit a low incidence of cancer. Data show Cr(VI) induces fewer chromosome aberrations in whale cells after acute Cr(VI) exposure suggesting whale cells can evade Cr(VI)-induced chromosome instability. However, it is unknown if whales can evade Cr(VI)-induced chromosome instability. Thus, we tested the hypothesis that whale cells resist Cr(VI)-induced loss of homologous recombination repair activity and increased SAC bypass and centrosome amplification. We found Cr(VI) induces similar amounts of DNA double-strand breaks after acute (24 h) and prolonged (120 h) exposures in whale lung cells, but does not inhibit homologous recombination repair, SAC bypass, or centrosome amplification, and does not induce chromosome instability. These data indicate whale lung cells resist Cr(VI)-induced chromosome instability, the major driver for Cr(VI) carcinogenesis at a cellular level, consistent with observations that whales are resistant to cancer.

We are facing a critical aging crisis, with geriatric populations (65+) growing to unprecedented proportions and ~4 million people (a 6.5-fold increase) expected to become centenarians by 2050. This is compounded by environmental pollution, which affects individuals of all ages and contributes to age-related diseases. As we have a limited understanding of how environmental pollutants affect older populations distinctly from younger populations, these longer-lived geriatric populations present a key knowledge gap. To address this knowledge gap, we employ a “Toxic Aging Coin” approach: heads consider how age impacts chemical toxicity, and tails consider how chemicals act as gerontogens—or how they accelerate biological aging. We employed this approach to investigate hexavalent chromium (Cr[VI]) impacts on female rats exposed to 0.05 or 0.1 mg Cr(VI)/L in drinking water for 90 days; these are the maximum contaminant levels (i.e., the highest levels permitted) from the World Health Organization and U.S. Environmental Protection Agency, respectively. During exposure, rats performed a battery of behavior assays to assess grip strength, locomotor coordination, anxiety, spatial memory, sociability, and social novelty preference. We observed age differences in Cr(VI) neurotoxicity, with grip strength, locomotor function, and spatial memory in middle-aged females being particularly affected. We further compared these results in females to results in males, noting many sex differences, especially in middle-aged rats. These data emphasize the need to consider age and sex as variables in toxicology and to revisit drinking water regulations for Cr(VI).

Hexavalent chromium (Cr[VI]) is a widespread environmental pollutant in air and water that is primarily attributed to industrial pollution. The current maximum contaminant levels (MCLs) for drinking water from the World Health Organization and the U.S. Environmental Protection Agency (0.05 and 0.1 mg/L, respectively) were set based on contact dermatitis and warrant further toxicological investigation. While Cr(VI) is neurotoxic and accumulates in the brain, most animal studies only report whole-brain Cr, leaving large knowledge gaps. Few studies consider differences between ages or sexes, and fewer consider essential metal dyshomeostasis. We sought to investigate where Cr accumulates in the brain, considering sex and age differences, following a 90-day drinking water exposure to current MCLs. Here, we report Cr levels in six brain regions of rats exposed to drinking water Cr(VI). We observed Cr only accumulated in the hippocampus, and only in older females. We further assessed changes to essential metals in the hippocampus, observing opposite effects across sexes and between young rats compared to older rats. In sum, our data indicate drinking water Cr(VI) selectively targeted the hippocampus, with geriatric females accumulating the most Cr, and induced significant essential metal dyshomeostasis even in tissues lacking evident Cr accumulation.

Hexavalent chromium [Cr(VI)] is an established human lung carcinogen, but the carcinogenesis mechanism is poorly understood. Chromosome instability, a hallmark of lung cancer, is considered a major driver of Cr(VI)-induced lung cancer. Unrepaired DNA double strand breaks are the underlying cause, and homologous recombination repair is the primary mechanism preventing Cr(VI)-induced DNA breaks from causing chromosome instability. Cell culture studies show acute Cr(VI) exposure causes DNA double strand breaks and increases homologous recombination repair activity. However, the ability of Cr(VI)-induced DNA breaks and repair impact has only been reported in cell culture studies. Therefore, we investigated whether acute Cr(VI) exposure could induce breaks and homologous recombination repair in rat lungs. Male and female Wistar rats were acutely exposed to either zinc chromate particles in a saline solution or saline alone by oropharyngeal aspiration. This exposure route resulted in increased Cr levels in each lobe of the lung. We found Cr(VI) induced DNA double-strand breaks in a concentration-dependent manner, with females being more susceptible than males, and induced homologous recombination repair at similar levels in both sexes. Thus, these data show this driving mechanism discovered in cell culture indeed translates to lung tissue in vivo.

We are facing a rapidly growing geriatric population (65+) that will live for multiple decades and are challenged with environmental pollution far exceeding that of previous generations. Consequently, we currently have a poor understanding of how environmental pollution will impact geriatric health distinctly from younger populations. Few toxicology studies have considered age differences with geriatric individuals. Critically, all top ten most prevalent age-related diseases are linked to metal exposures. Hexavalent chromium [Cr(VI)] is a metal of major environmental health concern that can induce aging phenotypes and neurotoxicity. However, there are many knowledge gaps for Cr(VI) neurotoxicity, including how Cr(VI) impacts behavior. To address this, we exposed male rats across three ages (3-, 7-, and 18-months old) to Cr(VI) in drinking water (0, 0.05, 0.1 mg/L) for 90 days. These levels reflect the maximum contaminant levels determined by the World Health Organization (WHO) and the U.S. Environmental Protection Agency (US EPA). Here, we report how these Cr(VI) drinking water levels impacted rat behaviors using a battery of behavior tests, including grip strength, open field assay, elevated plus maze, Y-maze, and 3-chamber assay. We observed adult rats were the most affected age group and memory assays (spatial and social) exhibited the most significant effects. Critically, the significant effects were surprising as rats should be particularly resistant to these Cr(VI) drinking water levels due to the adjustments applied in risk assessment from rodent studies to human safety, and because rats endogenously synthesize vitamin C in their livers (vitamin C is a primary reducer of Cr[VI] to Cr[III]). Our results emphasize the need to broaden the scope of toxicology research to consider multiple life stages and suggest the current regulations for Cr(VI) in drinking water need to be revisited.

Hexavalent chromium [Cr(VI)] is a human lung carcinogen with widespread exposure risks. Cr(VI) causes DNA double strand breaks that if unrepaired, progress into chromosomal instability (CIN), a key driving outcome in Cr(VI)-induced tumors. The ability of Cr(VI) to cause DNA breaks and inhibit repair is poorly understood in human lung epithelial cells, which are extremely relevant since pathology data show Cr(VI)-induced tumors originate from bronchial epithelial cells. In the present study, we considered immortalized and primary human bronchial epithelial cells. Cells were treated with zinc chromate at concentrations ranging 0.05 to 0.4μg/cm2 for acute (24 h) and prolonged (120 h) exposures. DNA double strand breaks (DSBs) were measured by neutral comet assay and the status of homologous recombination repair, the main pathway to fix Cr(VI)-induced DSBs, was measured by RAD51 foci formation with immunofluorescence, RAD51 localization with confocal microscopy and sister chromatid exchanges. We found acute and prolonged Cr(VI) exposure induced DSBs. Acute exposure induced homologous recombination repair, but prolonged exposure inhibited it resulting in chromosome instability in immortalized and primary human bronchial epithelial cells.

Chronic arsenic exposure through drinking water is a global health issue, affecting >200 million people. Arsenic is a group I human carcinogen and causes chromosomal instability (CIN). Arsenic exposure is the second most common cause of skin cancer after UV radiation. hsa-miR-186 is overexpressed in arsenic-induced squamous cell carcinoma relative to premalignant hyperkeratosis. Among predicted targets of hsa-miR-186 are cell cycle regulators including regulators of mitotic progression. Disruption of mitotic progression can contribute to CIN. Thus, we hypothesized that hsa-miR-186 overexpression contributes to malignant transformation of arsenic exposed HaCaT cells by induction of CIN. Stable clones of HaCaT cells transfected with pEP-hsa-miR-186 expression vector or empty vector were maintained under puromycin selection and exposed to 0 or 100 nM NaAsO2 and cultured for 29 weeks. HaCaT clones overexpressing hsa-miR-186 and exposed to NaAsO2 showed increased CIN and anchorage independent growth at 29 weeks in a stochastic manner, in contrast to unexposed empty vector transfected clones. These results suggest that clonal variability mediates arsenic-induced carcinogenesis in hsamiR-186 overexpressing human keratinocytes.

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical

Hexavalent chromium [Cr(VI)] is a well-known and widespread environmental contaminant associated with a variety of adverse health effects, in particular lung cancer. The primary route of exposure in humans is through inhalation. Particulate forms of Cr(VI) are the most potent but in vivo studies are difficult. Intratracheal instil-lation requires highly trained surgical procedures which also limits the number of repeated exposures possible and thus requires high doses. Inhalation studies can deliver lower more chronic doses but are expensive and generate dangerous aerosols. We evaluated an oropharyngeal aspiration exposure route for zinc chromate par-ticles in Wistar rats. Animals were treated once per week for 90 days. We found chromium accumulated in the lungs, blood, and reproductive tissues of all treated animals. Additionally, we found inflammatory indicators in the lung were elevated and circulating lymphocytes had increased chromosomal damage. These results show oropharyngeal aspiration provides a practicable exposure route for chronic and sub-chronic exposures of Cr(VI) particles.