Igor Koturbash

Germ-line mutations, DNA damage, and global hypermethylation in mice exposed to particulate air pollution in an urban/industrial location

Particulate air pollution is widespread, yet we have little understanding of the long-term health implications associated with exposure. We investigated DNA damage, mutation, and methylation in gametes of male mice exposed to particulate air pollution in an industrial/urban environment. C57BL/CBA mice were exposed in situ to ambient air near two integrated steel mills and a major highway, alongside control mice breathing high-efficiency air particulate (HEPA) filtered ambient air. PCR analysis of an expanded simple tandem repeat (ESTR) locus revealed a 1.6-fold increase in sperm mutation frequency in mice exposed to ambient air for 10 wks, followed by a 6-wk break, compared with HEPA-filtered air, indicating that mutations were induced in spermatogonial stem cells. DNA collected after 3 or 10 wks of exposure did not exhibit increased mutation frequency. Bulky DNA adducts were below the detection threshold in testes samples, suggesting that DNA reactive chemicals do not reach the germ line and cause ESTR mutation. In contrast, DNA strand breaks were elevated at 3 and 10 wks, possibly resulting from oxidative stress arising from exposure to particles and associated airborne pollutants. Sperm DNA was hypermethylated in mice breathing ambient relative to HEPA-filtered air and this change persisted following removal from the environmental exposure. Increased germ-line DNA mutation frequencies may cause population-level changes in genetic composition and disease. Changes in methylation can have widespread repercussions for chromatin structure, gene expression and genome stability. Potential health effects warrant extensive further investigation.

Fractionated Low-Dose Radiation Exposure Leads to Accumulation of DNA Damage and Profound Alterations in DNA and Histone Methylation in the Murine Thymus

Thymus, an important component of hematopoietic tissue, is a well-documented “target” of radiation carcinogenesis. Both acute and fractionated irradiation result in a high risk of leukemia and thymic lymphoma. However, the exact mechanisms underlying radiation-induced predisposition to leukemia and lymphoma are still unknown, and the contributions of genetic and epigenetic mechanisms in particular have yet to be defined. Global DNA hypomethylation is a well-known characteristic of cancer cells. Recent studies have also shown that tumor cells undergo prominent changes in histone methylation, particularly a substantial loss of trimethylation of histone H4-Lys20 and demethylation of genomic DNA. These losses are considered a universal marker of malignant transformation. In the present study, we investigated the effect of low-dose radiation exposure on the accumulation of DNA lesions and alterations of DNA methylation and histone H4-Lys20 trimethylation in the thymus tissue using an in vivo murine model. For the first time, we show that fractionated whole-body application of 0.5 Gy X-ray leads to decrease in histone H4-Lys20 trimethylation in the thymus. The loss of histone H4-Lys20 trimethylation was accompanied by a significant decrease in global DNA methylation as well as the accumulation of DNA damage as monitored by persistence of histone γH2AX foci in the thymus tissue of mice exposed to fractionated irradiation. Altered DNA methylation was associated with reduced expression of maintenance (DNMT1) and, to a lesser extent, de novo DNA methyltransferase DNMT3a in exposed animals. Expression of another de novo DNA methyltransferase DNMT3b was decreased only in males. Irradiation also resulted in ∼20% reduction in the levels of methyl-binding proteins MeCP2 and MBD2. Our results show the involvement of epigenetic alterations in radiation-induced responses in vivo. These changes may play a role in genome destabilization that ultimately leads to cancer.

Small molecules with big effects: The role of the microRNAome in cancer and carcinogenesis

Small non-coding RNAs-microRNAs, are potent negative regulators of gene expression. MicroRNAs are involved in multiple biological processes, metabolic regulation, including cell proliferation, differentiation, and programmed cell death. Since the dysregulation of these processes is a hallmark of cancer, microRNAs can be viewed as major contributors to the pathogenesis of cancer, including initiation and progression of cancer. This review focuses on microRNA biogenesis and function, and their role in cancer, metastasis, drug resistance, and tumorigenesis.

Radiation-induced bystander effects in vivo are epigenetically regulated in a tissue-specific manner.

Exposure of animal body parts to ionizing radiation (IR) can lead to molecular changes in distant shielded “bystander” tissues and organs. Nevertheless, tissue specificity of bystander responses within the same organism has not been examined in detail. Studies on in vivo bystander effect conducted so far analyzed changes induced by single‐dose exposure. The potential of fractionated irradiation to induce bystander effects in vivo has never been studied. We analyzed changes in global DNA methylation and microRNAome in skin and spleen of animals subjected to single‐dose (acute or fractionated) whole‐body or cranial exposure to 0.5 Gy of X‐rays. We found that IR‐induced DNA methylation changes in bystander spleen and skin were distinct. Acute radiation exposure resulted in a significant loss of global DNA methylation in the exposed and bystander spleen 6 hr, 96 hr, and 14 days after irradiation. Fractionated irradiation led to hypomethylation in bystander spleen 6 hr after whole‐body exposure, and 6 hr, 96 hr, and 14 days after cranial irradiation. Contrarily, changes in the skin of the same animals were seen only 6 hr after acute whole‐body and head exposure. DNA hypomethylation observed in spleen was paralleled by a reduction of methyl‐binding protein MeCP2 expression. Irradiation also induced tissue‐specific microRNAome alterations in skin and spleen. For the first time, we have shown that IR‐induced epigenetic bystander effects that occur in the same organism are triggered by both acute and fractionated exposure and are very distinct in different bystander organs. Future studies are clearly needed to address organismal and carcinogenic repercussions of those changes. Environ. Mol. Mutagen., 2009.

Hypomethylation and genome instability in the germline of exposed parents and their progeny is associated with altered miRNA expression

Recent studies suggest that transgenerational genome instability may be epigenetic in nature and mediated via altered DNA methylation and microRNAome. Here, we investigated the nature and mechanisms underlying the disruption of DNA methylation and microRNA expression status in the germline and progeny of exposed parents. We have found that paternal irradiation leads to upregulation of the miR-29 family in the exposed male germline, which causes decreased expression of de novo methyltransferase, DNA methyltransferase 3a, and profound hypomethylation of long interspersed nuclear elements 1 (LINE1) and short interspersed nuclear elements B2 (SINE B2). Epigenetic changes in the male germline further resulted in deleterious effects in the somatic thymus tissue from the progeny of exposed animals, including hypomethylation of LINE1 and SINE B2. Hypomethylation of LINE1 and SINE B2 in the thymus tissue from the progeny was associated with a significant decrease in the levels of lymphoid-specific helicase (LSH) that is crucial for the maintenance of methylation and silencing of repetitive elements. Furthermore, we noted a significant upregulation of miR-468 that targets LSH and leads to its decreased expression in thymus in the progeny of exposed parents. We suggest that miR-468-mediated suppression of LSH leads to aberrant methylation of LINE1 and SINE B2. In summary, altered microRNAome and hypomethylation of retroelements constitute deleterious effects that may significantly influence genome stability of the parental germline and consequently cause genome instability in the progeny.

Sex-specific radiation-induced microRNAome responses in the hippocampus, cerebellum and frontal cortex in a mouse model.

Ionizing radiation is an important treatment modality, but it is also a well-known genotoxic agent capable of damaging cells and tissues. Therefore radiation treatment can cause numerous side effects in exposed tissues and organs. Radiotherapy is a part of the front-line treatment regime for brain cancer patients, but can cause severe functional and morphological changes in exposed brain tissues. However, the mechanisms of radiation-induced effects in the brain are not well understood and are under-investigated. Recent data has implicated short RNAs, especially microRNAs, as important in radiation responses, yet nothing is known about radiation-induced changes in the brain microRNAome. We analyzed the effects of X-ray irradiation on microRNA expression in the hippocampus, frontal cortex, and cerebellum of male and female mice. Here, we report tissue-, time-, and sex-specific brain radiation responses, as well as show evidence of an interplay between microRNAs and their targets. Specifically, we show that changes in the expression of the miR-29 family may be linked, at least in part, to altered expression of de novo methyltransferase DNMT3a and changed global DNA methylation levels. Further, these sex-specific epigenetic changes may be correlated to the prevalence of radiation-induced cancers in males. We identified several microRNAs that can potentially serve as biomarkers of brain radiation exposure. In summary, our study may provide an important roadmap for further analysis of microRNA expression in different brain regions of male and female mice and for detailed dissection of radiation-induced brain responses.

Role of epigenetic events in chemical carcinogenesis--a justification for incorporating epigenetic evaluations in cancer risk assessment.

Recent advances in field of cancer research have established that all major human cancers, in addition to having a large number of genetic alterations, exhibit prominent epigenetic abnormalities that can be used as biomarkers for the molecular diagnosis of cancer. Currently, epigenetic markers have shown promise in establishing the diagnosis and prognosis of all major human cancers. Additionally, accumulating evidence suggests that epigenetic alterations may be early indicators of genotoxic and non-genotoxic carcinogenic exposure and may be used as biomarkers in the assessment of the carcinogenic potential of environmental chemical and physical agents. This review presents current evidence on the role of epigenetic alterations in chemical carcinogenesis and highlights a number of advantages of epigenetic biomarkers over traditionally used methods in cancer risk assessment.

Altered microRNA expression patterns in irradiated hematopoietic tissues suggest a sex-specific protective mechanism

To investigate involvement of miRNAs in radiation responses we used microRNAome profiling to analyze the sex-specific response of radiation sensitive hematopoietic lymphoid tissues. We show that radiation exposure resulted in a significant and sex-specific deregulation of microRNA expression in murine spleen and thymus tissues. Among the regulated miRNAs, we found that changes in expression of miR-34a and miR-7 may be involved in important protective mechanisms counteracting radiation cytotoxicity. We observed a significant increase in the expression of tumor-suppressor miR-34a, paralleled by a decrease in the expression of its target oncogenes NOTCH1, MYC, E2F3 and cyclin D1. Additionally, we show that miR-7 targets the lymphoid-specific helicase LSH, a pivotal regulator of DNA methylation and genome stability. While miR-7 was significantly down-regulated LSH was significantly up-regulated. These cellular changes may constitute an attempt to counteract radiation-induced hypomethylation. Tissue specificity of miRNA responses and possible regulation of miRNA expression upon irradiation are discussed.

Paternal cranial irradiation induces distant bystander DNA damage in the germline and leads to epigenetic alterations in the offspring.

It is now well accepted that parental whole body irradiation causes transgenerational genome and epigenome instability in the offspring. The majority of human exposures to radiation, such as therapeutic and diagnostic irradiation, are localized and focused. The potential of localized body-part exposures to affect the germline and thus induce deleterious changes in the progeny has not been studied. To investigate whether or not the paternal cranial irradiation can exert deleterious changes in the protected germline, we studied the accumulation of DNA damage in the shielded testes tissue. Here we report that the localized paternal cranial irradiation results in a significant accumulation of unrepaired DNA lesions in sperm cells and leads to a profound epigenetic dysregulation in the unexposed progeny conceived a week after paternal exposure.

Sex-specific microRNAome deregulation in the shielded bystander spleen of cranially exposed mice

The bystander effect is a phenomenon that occurs when exposed cells signal distress to their naïve, unexposed neighbors. It is now accepted as a ubiquitous consequence of radiation exposure. It is well documented to occur in cultured cells, 3D tissue models, and in organs and organisms. Notwithstanding, the exact mechanisms of the bystander effect remain unclear. Recent studies hinted that bystander effects may, in part, be distinct in males and females, and possibly mediated via short non-coding RNAs, specifically, microRNAs. MicroRNAs are small, abundant, and capable of regulating the expression of a wide variety of targets. Yet, their roles in bystander effects have not been analyzed in detail. The mechanisms behind sex differences observed in in vivo bystander effects also remain to be uncovered. We hypothesized that the radiation-induced expression of microRNAs in exposed and bystander tissue may be distinct in males and females. Using a well-establish bystander mouse model when the animal’s head is exposed, while the body is completely protected by a medical-grade shield, we have for the first time shown that radiation exposure triggers a significant and sex-specific deregulation of the microRNAome in the non-exposed bystander spleen. The altered miRNA levels were paralleled by sex-specific changes in the levels of the miRNA processing enzyme Dicer and components of the RNA-induced silencing complex (RISC). Sterilization of animals resulted in drastic microRNAome alterations and significantly affected radiation and bystander miRNA responses. Our data may provide a roadmap for further analysis of the role of microRNAome in genotoxic stress responses and may help us explain sex specificity of radiation-induced carcinogenesis.