Unexpected Enhancement of Cytotoxicity of Cisplatin in a Rat Kidney Proximal Tubular Cell Line Overexpressing Mitochondrial Glutathione Transport Activity.

In previous studies, we identified the two principal transporters that mediate the uptake of glutathione (GSH) from cytoplasm into the mitochondrial matrix of rat kidney proximal tubular cells. We hypothesized that genetic modulation of transporter expression could markedly alter susceptibility of renal proximal tubular cells to a broad array of oxidants and mitochondrial toxicants. Indeed, we previously showed that overexpression of either of these transporters resulted in diminished susceptibility to several chemicals. In the present work, we investigated the influence of overexpression of the mitochondrial 2-oxoglutarate carrier (OGC) in NRK-52E cells on the cytotoxicity of the antineoplastic drug cisplatin. In contrast to previous results showing that overexpression of the mitochondrial OGC provided substantial protection of NRK-52E cells from injury due to several toxicants, we found a remarkable enhancement of cellular injury from exposure to cisplatin as compared to wild-type NRK-52E cells. Despite the oxidative stress that cisplatin is known to cause in the renal proximal tubule, the increased concentrations of mitochondrial GSH associated with OGC overexpression likely resulted in increased delivery of cisplatin to molecular targets and increased cellular injury rather than the typical protection observed in the previous work.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate trichloroethylene reproductive toxicity via metabolism in Wistar rats.

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.

Diverse Roles of Mitochondria in Renal Injury from Environmental Toxicants and Therapeutic Drugs.

Mitochondria are well-known to function as the primary sites of ATP synthesis in most mammalian cells, including the renal proximal tubule. Other functions have also been associated with different mitochondrial activities, including the regulation of redox status and the initiation of mitophagy and apoptosis. Mechanisms for the membrane transport of glutathione (GSH) and various GSH-derived metabolites across the mitochondrial inner membrane of renal proximal tubular cells are critical determinants of these functions and may serve as pharmacological targets for potential therapeutic approaches. Specific interactions of reactive intermediates, derived from drug metabolism, with molecular components in mitochondria have been identified as early steps in diverse forms of chemically-induced nephrotoxicity. Applying this key observation, we developed a novel hypothesis regarding the identification of early, sensitive, and specific biomarkers of exposure to nephrotoxicants. The underlying concept is that upon exposure to a diverse array of environmental contaminants, as well as therapeutic drugs whose efficacy is limited by nephrotoxicity, renal mitochondria will release both high- and low-molecular-weight components into the urine or the extracellular medium in an in vitro model. The detection of these components may then serve as indicators of exposure before irreversible renal injury has occurred.

Target Organ Metabolism, Toxicity, and Mechanisms of Trichloroethylene and Perchloroethylene: Key Similarities, Differences, and Data Gaps.

Trichloroethylene (TCE) and perchloroethylene or tetrachloroethylene (PCE) are high-production volume chemicals with numerous industrial applications. As a consequence of their widespread use, these chemicals are ubiquitous environmental contaminants to which the general population is commonly exposed. It is widely assumed that TCE and PCE are toxicologically similar; both are simple olefins with three (TCE) or four (PCE) chlorines. Nonetheless, despite decades of research on the adverse health effects of TCE or PCE, few studies have directly compared these two toxicants. Although the metabolic pathways are qualitatively similar, quantitative differences in the flux and yield of metabolites exist. Recent human health assessments have uncovered some overlap in target organs that are affected by exposure to TCE or PCE, and divergent species- and sex-specificity with regard to cancer and noncancer hazards. The objective of this minireview is to highlight key similarities, differences, and data gaps in target organ metabolism and mechanism of toxicity. The main anticipated outcome of this review is to encourage research to 1) directly compare the responses to TCE and PCE using more sensitive biochemical techniques and robust statistical comparisons; 2) more closely examine interindividual variability in the relationship between toxicokinetics and toxicodynamics for TCE and PCE; 3) elucidate the effect of coexposure to these two toxicants; and 4) explore new mechanisms for target organ toxicity associated with TCE and/or PCE exposure.

Reactive Oxygen Stimulation of Interleukin-6 Release in the Human Trophoblast Cell Line HTR-8/SVneo by the Trichlorethylene Metabolite S-(1,2-Dichloro)-l-Cysteine.

Trichloroethylene (TCE) is a common environmental pollutant associated with adverse reproductive outcomes in humans. TCE intoxication occurs primarily through its biotransformation to bioactive metabolites, including S-(1,2-dichlorovinyl)-l-cysteine (DCVC). TCE induces oxidative stress and inflammation in the liver and kidney. Although the placenta is capable of xenobiotic metabolism and oxidative stress and inflammation in placenta have been associated with adverse pregnancy outcomes, TCE toxicity in the placenta remains poorly understood. We determined the effects of DCVC by using the human extravillous trophoblast cell line HTR-8/SVneo. Exposure to 10 and 20 µM DCVC for 10 h increased reactive oxygen species (ROS) as measured by carboxydichlorofluorescein fluorescence. Moreover, 10 and 20 µM DCVC increased mRNA expression and release of interleukin-6 (IL-6) after 24-h exposure, and these responses were inhibited by the cysteine conjugate beta-lyase inhibitor aminooxyacetic acid and by treatments with antioxidants (alpha-tocopherol and deferoxamine), suggesting that DCVC-stimulated IL-6 release in HTR-8/SVneo cells is dependent on beta-lyase metabolic activation and increased generation of ROS. HTR-8/SVneo cells exhibited decreased mitochondrial membrane potential at 5, 10, and 20 µM DCVC at 5, 10, and 24 h, showing that DCVC induces mitochondrial dysfunction in HTR-8/Svneo cells. The present study demonstrates that DCVC stimulated ROS generation in the human placental cell line HTR-8/SVneo and provides new evidence of mechanistic linkage between DCVC-stimulated ROS and increase in proinflammatory cytokine IL-6. Because abnormal activation of cytokines can disrupt trophoblast functions necessary for placental development and successful pregnancy, follow-up investigations relating these findings to physiologic outcomes are warranted.

Glutathione Levels and Susceptibility to Chemically Induced Injury in Two Human Prostate Cancer Cell Lines.

More aggressive prostate cancer cells (PCCs) are often resistant to chemotherapy. Differences exist in redox status and mitochondrial metabolism that may help explain this phenomenon. Two human PCC lines, PC-3 cells (more aggressive) and LNCaP cells (less aggressive), were compared with regard to cellular glutathione (GSH) levels, susceptibility to either oxidants or GSH depletors, and expression of several proteins involved in apoptosis and stress response to test the hypothesis that more aggressive PCCs exhibit higher GSH concentrations and are relatively resistant to cytotoxicity. PC-3 cells exhibited 4.2-fold higher GSH concentration than LNCaP cells but only modest differences in acute cytotoxicity were observed at certain time points. However, only LNCaP cells underwent diamide-induced apoptosis. PC-3 cells exhibited higher levels of Bax and caspase-8 cleavage product but lower levels of Bcl-2 than LNCaP cells. However, LNCaP cells exhibited higher expression of Fas receptor (FasR) but also higher levels of several stress response and antioxidant proteins than PC-3 cells. LNCaP cells also exhibited higher levels of several mitochondrial antioxidant systems, suggesting a compensatory response. Thus, significant differences in redox status and expression of proteins involved in apoptosis and stress response may contribute to PCC aggressiveness.

Multigenerational study of chemically induced cytotoxicity and proliferation in cultures of human proximal tubular cells.

Primary cultures of human proximal tubular (hPT) cells are a useful experimental model to study transport, metabolism, cytotoxicity, and effects on gene expression of a diverse array of drugs and environmental chemicals because they are derived directly from the in vivo human kidney. To extend the model to investigate longer-term processes, primary cultures (P0) were passaged for up to four generations (P1-P4). hPT cells retained epithelial morphology and stained positively for cytokeratins through P4, although cell growth and proliferation successively slowed with each passage. Necrotic cell death due to the model oxidants tert-butyl hydroperoxide (tBH) and methyl vinyl ketone (MVK) increased with increasing passage number, whereas that due to the selective nephrotoxicant S-(1,2-dichlorovinyl)-l-cysteine (DCVC) was modest and did not change with passage number. Mitochondrial activity was lower in P2-P4 cells than in either P0 or P1 cells. P1 and P2 cells were most sensitive to DCVC-induced apoptosis. DCVC also increased cell proliferation most prominently in P1 and P2 cells. Modest differences with respect to passage number and response to DCVC exposure were observed in expression of three key proteins (Hsp27, GADD153, p53) involved in stress response. Hence, although there are some modest differences in function with passage, these results support the use of multiple generations of hPT cells as an experimental model.

Renal Glutathione: Dual roles as antioxidant protector and bioactivation promoter.

The tripeptide glutathione (GSH) possesses two key structural features, namely the nucleophilic sulfur and the γ-glutamyl isopeptide bond. The former allows GSH to serve as a critical antioxidant and anti-electrophile. The latter allows GSH to translocate throughout the systemic circulation without being degraded. The kidneys exhibit several unique processes for handling GSH. This includes the extraction of 80% of plasma GSH, in part by glomerular filtration but mostly by transport across the basolateral plasma membrane. Studies on the protective effect of exogenous GSH are summarized, showing the different inherent susceptibility of proximal tubular and distal tubular cells and the impact on pathological or disease states, including hypoxia, diabetic nephropathy, and compensatory renal growth associated with uninephrectomy. Studies on mitochondrial GSH transport show the coordination between the citric acid cycle and oxidative phosphorylation in generating driving forces for both plasma membrane and mitochondrial carriers. The strong protective effects of increasing expression and activity of these carriers against oxidants and mitochondrial toxicants are summarized. Although GSH plays a cytoprotective role in most situations, two distinct exceptions to this are presented. In contrast to expectations, overexpression of the mitochondrial 2-oxoglutarate carrier markedly increased cell death from exposure to the nephrotoxic chemotherapeutic drug cisplatin (CDDP). Another key example of GSH serving a bioactivation role in the kidneys, rather than a detoxification role, is the metabolism of halogenated alkenes such as trichloroethylene (TCE). Although considerable research has gone into this topic, unanswered questions and emerging topics remain and are discussed.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate toxicity of the trichloroethylene metabolite S-(1,2-dichlorovinyl)-L-cysteine in human placental villous trophoblast BeWo cells.

Trichloroethylene (TCE) is a known human carcinogen with toxicity attributed to its metabolism. S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is a metabolite of TCE formed downstream in TCE glutathione (GSH) conjugation and is upstream of several toxic metabolites. Despite knowledge that DCVC stimulates reactive oxygen species (ROS) generation and apoptosis in placental cells, the extent to which these outcomes are attributable to DCVC metabolism is unknown. The current study used N-acetyl-L-cysteine (NAC) at 5 mM and aminooxyacetic acid (AOAA) at 1 mM as pharmacological modifiers of DCVC metabolism to investigate DCVC toxicity at concentrations of 5-50 µM in the human placental trophoblast BeWo cell model capable of forskolin-stimulated syncytialization. Exposures of unsyncytialized BeWo cells, BeWo cells undergoing syncytialization, and syncytialized BeWo cells were studied. NAC pre/co-treatment with DCVC either failed to inhibit or exacerbated DCVC-induced H2O2 abundance, PRDX2 mRNA expression, and BCL2 mRNA expression. Although NAC increased mRNA expression of CYP3A4, which would be consistent with increased generation of the toxic metabolite N-acetyl-DCVC sulfoxide (NAcDCVCS), a CYP3A4 inhibitor ketoconazole did not significantly alter BeWo cell responses. Moreover, AOAA failed to inhibit cysteine conjugate ß-lyase (CCBL), which bioactivates DCVC, and did not affect the percentage of nuclei condensed or fragmented, a measure of apoptosis, in all BeWo cell models. However, syncytialized cells had higher CCBL activity compared to unsyncytialized cells, suggesting that the former may be more sensitive to DCVC toxicity. Together, although neither NAC nor AOAA mitigated DCVC toxicity, differences in CCBL activity and potentially CYP3A4 expression dictated the differential toxicity derived from DCVC.

Naturally occurring autoimmune disease in (NZB X NZW) F1 mice is correlated with suppression of MZ B cell development due to aberrant B Cell Receptor (BCR) signaling, which is exacerbated by exposure to inorganic mercury.

Autoimmune diseases are multifactorial and include environmental as well as genetic drivers. Although much progress has been made in understanding the nature of genetic underpinnings of autoimmune disease, by comparison much less is understood regarding how environmental factors interact with genetics in the development of autoimmunity and autoimmune disease. In this report, we utilize the (NZB X NZW) F1 mouse model of Systemic Lupus Erythematosus (SLE). Mercury is a xenobiotic that is environmentally ubiquitous and is epidemiologically linked with the development of autoimmunity. Among other attributes of human SLE, (NZB X NZW) F1 mice spontaneously develop autoimmune-mediated kidney disease. It has been previously shown that if (NZB X NZW) F1 mice are exposed to inorganic mercury (Hg2+), the development of autoimmunity, including autoimmune kidney pathology, is accelerated. We now show that in these mice the development of kidney disease is correlated with a decreased percentage of marginal zone (MZ) B cells in the spleen. In Hg2+-intoxicated mice, kidney disease is significantly augmented, and matched by a greater decrease in MZ B cell splenic percentages than found in control mice. In Hg2+- intoxicated mice, the decrease in MZ B cells appears to be linked to aberrant B Cell Receptor (BCR) signal strength in transitory 2 (T2) B cells, developmental precursors of MZ B cells.

Adaptive changes in renal mitochondrial redox status in diabetic nephropathy.

Nephropathy is a serious and common complication of diabetes. In the streptozotocin (STZ)-treated rat model of diabetes, nephropathy does not typically develop until 30 to 45days post-injection, although hyperglycemia occurs within 24h. We tested the hypothesis that chronic hyperglycemia results in a modest degree of oxidative stress that is accompanied by compensatory changes in certain antioxidants and mitochondrial redox status. We propose that as kidneys progress to a state of diabetic nephropathy, further adaptations occur in mitochondrial redox status. Basic parameters of renal function in vivo and several parameters of mitochondrial function and glutathione (GSH) and redox status in isolated renal cortical mitochondria from STZ-treated and age-matched control rats were examined at 30days and 90days post-injection. While there was no effect of diabetes on blood urea nitrogen, measurement of other, more sensitive parameters, such as urinary albumin and protein, and histopathology showed significant and progressive worsening in diabetic rats. Thus, renal function is compromised even prior to the onset of frank nephropathy. Changes in mitochondrial respiration and enzyme activities indicated existence of a hypermetabolic state. Higher mitochondrial GSH content and rates of GSH transport into mitochondria in kidneys from diabetic rats were only partially due to changes in expression of mitochondrial GSH carriers and were mostly due to higher substrate supply. Although there are few clear indicators of oxidative stress, there are several redox changes that occur early and change further as nephropathy progresses, highlighting the complexity of the disease.

Cellular and Functional Biomarkers of Renal Injury and Disease.

Biomarkers, defined as molecules in biological samples that are used as indicators of organ function, can assess exposure to potentially injurious chemicals, effects on organ function, or susceptibility to organ functional decline. The kidneys are frequently exposed to many drugs and chemicals and loss of kidney function is a frequent consequence of diseases such as diabetes. This review summarizes findings reported in 2021 and early-2022 from clinical and experimental animal studies on biomarkers, focusing on five topics: 1) Progression and severity of diabetic kidney disease; 2) acute kidney injury (AKI) and chronic kidney disease (CKD) severity and prognosis; 3) progression of AKI to CKD; 4) renal cell carcinoma (RCC) severity and prognosis; and 5) detection of exposure to environmental chemicals and nephrotoxic drugs.

Placenta as a target of trichloroethylene toxicity.

Trichloroethylene (TCE) is an industrial solvent and a common environmental contaminant detected in thousands of hazardous waste sites. Risk of exposure is a concern for workers in occupations that use TCE as well as for residents who live near industries that use TCE or who live near TCE-contaminated sites. Although renal, hepatic and carcinogenic effects of TCE have been documented, less is known about TCE impacts on reproductive functions despite epidemiology reports associating maternal TCE exposure with adverse pregnancy outcomes. Toxicological evidence suggests that the placenta mediates at least some of the adverse pregnancy outcomes associated with TCE exposure. Toxicology studies show that the TCE metabolite, S-(1,2-dichlorovinyl)-l-cysteine (DCVC) generates toxic effects such as mitochondrial dysfunction, apoptosis, oxidative stress, and release of prostaglandins and pro-inflammatory cytokines in placental cell lines. Each of these mechanisms of toxicity have significant implications for placental functions and, thus, ultimately the health of mother and developing child. Despite these findings there remain significant gaps in our knowledge about effects of TCE on the placenta, including effects on specific placental cell types and functions as well as sex differences in response to TCE exposure. Due to the critical role that the placenta plays in pregnancy, future research addressing some of these knowledge gaps could lead to significant gains in public health.

Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury.

Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-l-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

Assessment of the renal toxicity of novel anti-inflammatory compounds using cynomolgus monkey and human kidney cells.

PF1, an anti-inflammatory drug candidate, was nephrotoxic in cynomolgus monkeys in a manner that was qualitatively comparable to that observed with the two previous exploratory drug candidates (PF2and PF3). Based on the severity of nephrotoxicity, PF1 ranked between the other two compounds, withPF2 inducing mortality at all doses and PF3 eliciting only mild nephrotoxicity. To further characterize nephrotoxicity in monkeys and enable direct comparisons with humans, primary cultures of proximal tubular (PT) cells from monkey and human kidneys were used as in vitro tools, using lactate dehydrogenase release as the biomarker of cytotoxicity. In both human and monkey PT cells, PF2was by far the most cytotoxic compound of the three drugs. PF1 exhibited modest cytotoxicity at the highest concentration tested in human PT cells but none in monkey kidney cells whereas PF3 exhibited the reverse pattern.Because these drugs are organic anions, mechanistic studies using human organic anion transporters 1 and 3 (hOAT1 andhOAT3) transfected cell lines were pursued to evaluate the potential of these compounds to interact with these transporters. All three drugs exhibited high affinity for hOAT3 (PF1 exhibited the lowest IC50 of 6M) but only weakly interacted with hOAT1 (with no interaction found for PF2). PF2 was a strong hOAT3 (not hOAT1) substrate, whereas PF1 and PF3 were substrates for both hOAT1 and hOAT3.Upon pretreatment of monkeys with the OAT substrate probenecid, PF3 systemic exposure (AUC) and half-life (t1/2) increased approximately 2-fold whereas clearance (CL) and volume of distribution (Vdss) decreased, as compared to naïve monkeys. This indicated that PF3 competed with probenecid for hOAT1 and/or hOAT3mediated elimination of PF3. Thus, hOAT1 and/or hOAT3 may be responsible for the uptake of this series of drugs in renal PT cells, which may directly or indirectly lead to the observed nephrotoxicity in vivo.

Environmental and Genetic Factors Influencing Kidney Toxicity.

The kidneys are a frequent target organ for toxicity from exposures to various environmental chemicals and agents. To understand the risk to human health from such exposures, it is important to consider both the underlying chemical and pathologic mechanisms and factors that may modify susceptibility to injury. Choices of exemplary environmental agents to review are based on those with selective effects on the kidneys and for which significant amounts of mechanistic and human data are available. These include the heavy metals cadmium and arsenic, fluoride, the organic solvents trichloroethylene and perchloroethylene, drinking water disinfection by-products haloacids, food and herbal drug contaminants aristolochic acid and melamine, and heat stress. Some common mechanistic features of all these diverse exposures are highlighted, and include oxidative stress and mitochondrial damage. Two major genetic factors that are discussed include genetic polymorphisms in plasma membrane transporters that catalyze uptake and accumulation or efflux and elimination of environmental chemicals, and genetic polymorphisms in bioactivation enzymes that generate toxic and reactive metabolites. Identification of methods to prevent environmental toxicant-associated kidney damage and understanding the genetic factors that influence kidney function and the kidney's response to exposures can be applied to refine risk assessments.

Trichloroethylene exposure in mid-pregnancy decreased fetal weight and increased placental markers of oxidative stress in rats.

Although epidemiology studies have associated maternal trichloroethylene (TCE) exposure with decreased birth weight and preterm birth, mechanistic explanations for these associations are currently lacking. We hypothesized that TCE targets the placenta with adverse consequences for pregnancy outcomes. Pregnant Wistar rats were exposed orally to vehicle or 480 mg TCE/kg body weight from gestational days (gd) 6-16, and tissues were collected on gd 16. Exposure to TCE significantly decreased average fetal weight without reducing maternal weight. In placenta, TCE significantly increased 8-hydroxy-deoxyguanosine, global 5-hydroxymethylcytosine, and mRNA expression of Tet3, which codes for an enzyme involved in 5-hydroxymethylcytosine formation. Furthermore, glutathione S-transferase activity and immunohistochemical staining were increased in placentas of TCE-exposed rats. The present study provides the first evidence that TCE increases markers of oxidative stress in placenta in a fetal growth restriction rat model, providing new insight into the placenta as a potentially relevant target for TCE-induced adverse pregnancy outcomes.

Bromate-induced Changes in p21 DNA Methylation and Histone Acetylation in Renal Cells.

Bromate (BrO3-) is a water disinfection byproduct (DBP) previously shown to induce nephrotoxicity in vitro and in vivo. We recently showed that inhibitors of DNA methyltransferase 5-aza-2'-deoxycytidine (5-Aza) and histone deacetylase trichostatin A (TSA) increased BrO3- nephrotoxicity whereas altering the expression of the cyclin-dependent kinase inhibitor p21. Human embryonic kidney cells (HEK293) and normal rat kidney (NRK) cells were sub-chronically exposed to BrO3- or epigenetic inhibitors for 18 days, followed by 9 days of withdrawal. DNA methylation was studied using a modification of bisulfite amplicon sequencing called targeted gene bisulfite sequencing. Basal promoter methylation in the human p21 promoter region was substantially lower than that of the rat DNA. Furthermore, 5-Aza decreased DNA methylation in HEK293 cells at the sis-inducible element at 3 distinct CpG sites located at 691, 855, and 895 bp upstream of transcription start site (TSS). 5-Aza also decreased methylation at the rat p21 promoter about 250 bp upstream of the p21 TSS. In contrast, sub-chronic BrO3- exposure failed to alter methylation in human or rat renal cells. BrO3- exposure altered histone acetylation in NRK cells at the p21 TSS, but not in HEK293 cells. Interestingly, changes in DNA methylation induced by 5-Aza persisted after its removal; however, TSA- and BrO3--induced histone hyperacetylation returned to basal levels after 3 days of withdrawal. These data demonstrate novel sites within the p21 gene that are epigenetically regulated and further show that significant differences exist in the epigenetic landscape between rat and human p21, especially with regards to toxicant-induced changes in histone acetylation.

Adapterama II: universal amplicon sequencing on Illumina platforms (TaggiMatrix).

Next-generation sequencing (NGS) of amplicons is used in a wide variety of contexts. In many cases, NGS amplicon sequencing remains overly expensive and inflexible, with library preparation strategies relying upon the fusion of locus-specific primers to full-length adapter sequences with a single identifying sequence or ligating adapters onto PCR products. In Adapterama I, we presented universal stubs and primers to produce thousands of unique index combinations and a modifiable system for incorporating them into Illumina libraries. Here, we describe multiple ways to use the Adapterama system and other approaches for amplicon sequencing on Illumina instruments. In the variant we use most frequently for large-scale projects, we fuse partial adapter sequences (TruSeq or Nextera) onto the 5' end of locus-specific PCR primers with variable-length tag sequences between the adapter and locus-specific sequences. These fusion primers can be used combinatorially to amplify samples within a 96-well plate (8 forward primers + 12 reverse primers yield 8 × 12 = 96 combinations), and the resulting amplicons can be pooled. The initial PCR products then serve as template for a second round of PCR with dual-indexed iTru or iNext primers (also used combinatorially) to make full-length libraries. The resulting quadruple-indexed amplicons have diversity at most base positions and can be pooled with any standard Illumina library for sequencing. The number of sequencing reads from the amplicon pools can be adjusted, facilitating deep sequencing when required or reducing sequencing costs per sample to an economically trivial amount when deep coverage is not needed. We demonstrate the utility and versatility of our approaches with results from six projects using different implementations of our protocols. Thus, we show that these methods facilitate amplicon library construction for Illumina instruments at reduced cost with increased flexibility. A simple web page to design fusion primers compatible with iTru primers is available at: http://baddna.uga.edu/tools-taggi.html. A fast and easy to use program to demultiplex amplicon pools with internal indexes is available at: https://github.com/lefeverde/Mr_Demuxy.