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Although SARS-CoV-2 can cause severe illness and death, a percentage of the infected population is asymptomatic. This, along with other factors, such as insufficient diagnostic testing and underreporting due to self-testing, contributes to the silent transmission of SARS-CoV-2 and highlights the importance of implementing additional surveillance tools. The fecal shedding of the virus from infected individuals enables its detection in community wastewater, and this has become a valuable public health tool worldwide as it allows the monitoring of the disease on a populational scale. Here, we monitored the presence of SARS-CoV-2 and its dynamic genomic changes in wastewater sampled from two metropolitan areas in Arkansas during major surges of COVID-19 cases and assessed how the viral titers in these samples related to the clinical case counts between late April 2020 and January 2022. The levels of SARS-CoV-2 RNA were quantified by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) using a set of TaqMan assays targeting three different viral genes (encoding ORF1ab polyprotein, surface glycoprotein, and nucleocapsid phosphoprotein). An allele-specific RT-qPCR approach was used to screen the samples for SARS-CoV-2 mutations. The identity and genetic diversity of the virus were further investigated through amplicon-based RNA sequencing, and SARS-CoV-2 variants of concern were detected in wastewater samples throughout the duration of this study. Our data show how changes in the virus genome can affect the sensitivity of specific RT-qPCR assays used in COVID-19 testing with the surge of new variants. A significant association was observed between viral titers in wastewater and recorded number of COVID-19 cases in the areas studied, except when assays failed to detect targets due to the presence of particular variants. These findings support the use of wastewater surveillance as a reliable complementary tool for monitoring SARS-CoV-2 and its genetic variants at the community level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

It is well established that genotoxic reactivity of chemical carcinogens or their metabolites is a critical event in the initiation of tumorigenesis. However, the underlying mechanisms of events following initiation are less well understood, and with respect to genotoxic liver carcinogenesis, it is largely unknown how the initiated cells progress to form preneoplastic hepatic foci. In the present study, we investigated the underlying events associated with tumor-promoting activity of 2-acetylaminofluorene (2-AAF), a powerful complete genotoxic rat liver carcinogen. Male Sprague-Dawley rats were fed NIH-31 diet containing 0.02% of 2-AAF for 24 weeks, and the status of cytosine DNA methylation, histone methylation, and microRNA expression was determined in the livers of control and 2-AAF-fed rats. The results demonstrate that stages of multistage carcinogenesis following the initiation are driven primarily by carcinogen-induced epigenetic alterations. This was evidenced by altered global histone lysine methylation patterns, increased histone H3 lysine 9 and histone H3 lysine 27 trimethylation in the promoter regions of Rassf1a, p16(INK4a), Socs1, Cdh1, and Cx26 tumor suppressor genes, early Rassf1a and p16(INK4a) promoter CpG island hypermethylation, and altered microRNA expression in preneoplastic livers of rats exposed to 2-AAF. These changes were accompanied by dysregulation of the balance between cell proliferation and apoptosis, a fundamental pro-tumorigenic event in hepatocarcinogenesis. These results signify the fundamental role of epigenetic alterations in genotoxic liver carcinogenesis., (Published by Elsevier B.V.)

Background/aims: The importance of epigenetic changes in etiology and pathogenesis of disease has been increasingly recognized. However, the role of epigenetic alterations in the genesis of hepatic steatosis and cause of individual susceptibilities to this pathological state are largely unknown., Methods: Male inbred C57BL/6J and DBA/2J mice were fed a lipogenic methyl-deficient diet (MDD) that causes liver injury similar to human non-alcoholic steatohepatitis (NASH) for 6, 12, or 18 weeks, and the status of global and repetitive elements cytosine methylation, histone modifications, and expression of proteins responsible for those epigenetic modifications in livers was determined., Results: The development of hepatic steatosis in inbred C57BL/6J and DBA/2J mice was accompanied by prominent epigenetic abnormalities. This was evidenced by pronounced loss of genomic and repetitive sequences cytosine methylation, especially at major and minor satellites, accompanied by increased levels of repeat-associated transcripts, aberrant histone modifications, and alterations in expression of the maintenance DNA methyltransferase 1 (DNMT1) and de novo DNMT3A proteins in the livers of both mouse strains. However, the DBA/2J mice, which were characterized by an initially lower degree of methylation of repetitive elements and lower extent of histone H3 lysine 9 (H3K9) and H3 lysine 27 (H3K27) trimethylation in the normal livers, as compared to those in the C57BL/6J mice, developed more prominent NASH-specific pathomorphological changes., Conclusions: These results mechanistically link epigenetic alterations to the pathogenesis of hepatic steatosis and strongly suggest that differences in the cellular epigenetic status may be a predetermining factor to individual susceptibilities to hepatic steatosis.

Genomic hypomethylation is a consistent finding in both human and animal tumors and mounting experimental evidence suggests a key role for epigenetic events in tumorigenesis. Furthermore, it has been suggested that early changes in DNA methylation and histone modifications may serve as sensitive predictive markers in animal testing for carcinogenic potency of environmental agents. Alterations in metabolism of methyl donors, disturbances in activity and/or expression of DNA methyltransferases, and presence of DNA single-strand breaks could contribute to the loss of cytosine methylation during carcinogenesis; however, the precise mechanisms of genomic hypomethylation induced by chemical carcinogens remain largely unknown. This study examined the mechanism of DNA hypomethylation during hepatocarcinogenesis induced by peroxisome proliferators WY-14,643 (4-chloro-6-(2,3-xylidino)-pyrimidynylthioacetic acid) and DEHP (di-(2-ethylhexyl)phthalate), agents acting through non-genotoxic mode of action. In the liver of male Fisher 344 rats exposed to WY-14,643 (0.1% (w/w), 5 months), the level of genomic hypomethylation increased by approximately 2-fold, as compared to age-matched controls, while in the DEHP group (1.2% (w/w), 5 months) DNA methylation did not change. Global DNA hypomethylation in livers from WY-14,643 group was accompanied by the accumulation of DNA single-strand breaks, increased cell proliferation, and diminished expression of DNA methyltransferase 1, while the metabolism of methyl donors was not affected. In contrast, none of these parameters changed significantly in rats fed DEHP. Since WY-14,643 is much more potent carcinogen than DEHP, we conclude that the extent of loss of DNA methylation may be related to the carcinogenic potential of the chemical agent, and that accumulation of DNA single-strand breaks coupled to the increase in cell proliferation and altered DNA methyltransferase expression may explain genomic hypomethylation during peroxisome proliferator-induced carcinogenesis.

Peroxisome proliferators are potent rodent liver carcinogens that act via a non-genotoxic mechanism. The mode of action of these agents in rodent liver includes increased cell proliferation, decreased apoptosis, secondary oxidative stress and other events; however, it is not well understood how peroxisome proliferators are triggering the plethora of the molecular signals leading to cancer. Epigenetic changes have been implicated in the mechanism of liver carcinogenesis by a number of environmental agents. Short-term treatment with peroxisome proliferators and other non-genotoxic carcinogens leads to global and locus-specific DNA hypomethylation in mouse liver, events that were suggested to correlate with a burst of cell proliferation. In the current study, we investigated the effects of long-term exposure to a model peroxisome proliferator WY-14,643 on DNA and histone methylation. Male SV129mice were fed a control or WY-14,643-containing (1000ppm) diet for one week, five weeks or five months. Treatment with WY-14,643 led to progressive global hypomethylation of liver DNA as determined by an HpaII-based cytosine extension assay with the maximum effect reaching over 200% at five months. Likewise, trimethylation of histone H4 lysine 20 and H3 lysine 9 was significantly decreased at all time points. The majority of cytosine methylation in mammals resides in repetitive DNA sequences. In view of this, we measured the effect of WY-14,643 on the methylation status of major and minor satellites, as well as in IAP, LINE1 and LINE2 elements in liver DNA. Exposure to WY-14,643 resulted in a gradual loss of cytosine methylation in major and minor satellites, IAP, LINE1 and LINE2 elements. The epigenetic changes correlated with the temporal effects of WY-14,643 on cell proliferation rates in liver, but no sustained effect on c-Myc promoter methylation was observed. Finally, WY-14,643 had no effect on DNA and histone methylation status in Pparalpha-null mice at any of the time points considered in this study. These data indicate the importance of epigenetic alterations in the mechanism of action of peroxisome proliferators and the key role of Pparalpha.

Micro RNAs (miRNAs) are small non-coding RNA molecules that function as negative regulators of gene expression. They play a crucial role in the regulation of genes involved in the control of development, cell proliferation, apoptosis, and stress response. Although miRNA levels are substantially altered in tumors, their role in carcinogenesis, specifically at the early pre-cancerous stages, has not been established. Here we report that exposure of Fisher 344 rats to tamoxifen, a potent hepatocarcinogen in rats, for 24 weeks leads to substantial changes in the expression of miRNA genes in the liver. We noted a significant up-regulation of known oncogenic miRNAs, such as the 17-92 cluster, miR-106a, and miR-34. Furthermore, we confirmed the corresponding changes in the expression of proteins targeted by these miRNAs, which include important cell cycle regulators, chromatin modifiers, and expression regulators implicated in carcinogenesis. All these miRNA changes correspond to previously reported alterations in full-fledged tumors, including hepatocellular carcinomas. Thus, our findings indicate that miRNA changes occur prior to tumor formation and are not merely a consequence of a transformed state.

The methyl-deficient model of endogenous hepatocarcinogenesis in rodents is unique in that dietary omission rather than the addition of chemical carcinogens leads to tumor formation. Thus, the biochemical and molecular events predisposing to cancer in this model result from chronic metabolic stress and provide an ideal model system to study progressive alterations that occur during carcinogenesis. Moreover, epigenetic alterations imposed by this diet are believed to be 1 of the main mechanisms responsible for malignant transformation of rat liver cells. In this study we examined the changes in global histone modification patterns in liver during hepatocarcinogenesis induced by methyl deficiency. Feeding animals the methyl-deficient diet (MDD) led to progressive loss of histone H4 lysine 20 trimethylation (H4K20me3), H3 lysine 9 trimethylation (H3K9me3), and histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation. A considerable decrease of H4K20me3 and H3K9ac was also detected in liver tumors induced by MDD. In contrast, liver tumors displayed an increase in H3K9me3 and H4K16ac. To determine the possible mechanism of alterations of histone modifications, we analyzed the expression of histone-modifying enzymes in liver during hepatocarcinogenesis. The expression of Suv4-20h2 and RIZ1 histone methyltransferases (HMTs) steadily decreased along with the development of liver tumors and reached its lowest level in tumor tissue, whereas the expression of Suv39-h1 HMT and histone acetyltransferase 1 (HAT1) substantially increased in tumors. These results illustrate the complexity and importance of histone modification changes in the etiology of hepatocarcinogenesis induced by MDD.

Dietary methyl group deprivation is now well recognized as a model of hepatocarcinogenesis in rodents. In the present study, we examined the effects of feeding a methyl-deficient diet followed by a methyl-adequate diet on the extent of methylation of liver DNA and on the formation and evolution of altered hepatic foci. Male F344 rats were fed a methyl-deficient diet for 9, 18, 24, and 36 weeks, followed by re-feeding a methyl-adequate diet for a total of 54 weeks. Similar to previous findings, the methyl-deficient diet resulted in decreased levels of S-adenosylmethionine (SAM), SAM/SAH ratios, and global DNA hypomethylation. Feeding the methyl-adequate diet restored the liver SAM levels and SAM/SAH ratios to control levels in all experimental groups. In contrast, re-feeding the complete diet restored DNA methylation to normal level only in the group that had been fed the methyl-deficient diet for 9 weeks; in animals exposed to methyl deprivation longer, the methyl-adequate diet failed to reverse the hypomethylation of DNA. Liver tissue of rats exposed to methyl deficiency for 9, 18, 24, or 36 weeks was characterized by the persistent presence of placental isoform of glutathione-S-transferase (GSTpi)-positive lesions despite re-feeding the methyl-adequate diet. The persistence of altered hepatic foci in liver after withdrawal of methyl-deficient diet serves as an indication of the carcinogenic potential of a methyl-deficient diet. Substitution of the methyl-deficient diet with complete diet failed to prevent the expansion of initiated foci and restore DNA methylation in animals exposed to deficiency for 18, 24, or 36 weeks. The association between DNA hypomethylation and expansion of foci suggests that stable DNA hypomethylation is a promoting factor for clonal expansion of initiated cells. These results provide an experimental evidence and a mechanistic basis by which epigenetic alterations may contribute to the initiation and promotion steps of carcinogenesis.