Expression of Ceramide Synthases in Mice and Their Roles in Regulating Acyl-Chain Sphingolipids: A Framework for Baseline Levels and Future Implications in Aging and Disease.

Sphingolipids are an important class of lipids present in all eukaryotic cells that regulate critical cellular processes. Disturbances in sphingolipid homeostasis have been linked to several diseases in humans. Ceramides are central in sphingolipid metabolism and are largely synthesized by six ceramide synthase (CerS) isoforms (CerS1-6), each with a preference for different fatty acyl chain lengths. Although the tissue distribution of CerS mRNA expression in humans and the roles of CerS isoforms in synthesizing ceramides with different acyl chain lengths are known, it is unknown how CerS expression dictates ceramides and downstream metabolites within tissues. In this study, we analyzed sphingolipid levels and CerS mRNA expression in 3-month-old C57BL/6J mouse brain, heart, kidney, liver, lung, and skeletal muscle. The results showed that CerS expression and sphingolipid species abundance varied by tissue and that CerS expression was a predictor of ceramide species within tissues. Interestingly, although CerS expression was not predictive of complex sphingolipid species within all tissues, composite scores for CerSs contributions to total sphingolipids measured in each tissue correlated to CerS expression. Lastly, we determined that the most abundant ceramide species in mouse tissues aligned with CerS mRNA expression in corresponding human tissues (based on chain length preference), suggesting that mice are relevant preclinical models for ceramide and sphingolipid research. SIGNIFICANCE STATEMENT: The current study demonstrates that ceramide synthase (CerS) expression in specific tissues correlates not only with ceramide species but contributes to the generation of complex sphingolipids as well. As many of the CerSs and/or specific ceramide species have been implicated in disease, these studies suggest the potential for CerSs as therapeutic targets and the use of sphingolipid species as diagnostics in specific tissues.

The effect of the rs1799931 G857A (G286E) polymorphism on N-acetyltransferase 2-mediated carcinogen metabolism and genotoxicity differs with heterocyclic amine exposure.

Human N-acetyltransferase 2 (NAT2) is subject to genetic polymorphism in human populations. In addition to the reference NAT2*4 allele, two genetic variant alleles (NAT2*5B and NAT2*7B) are common in Europe and Asia, respectively. NAT2*5B possesses a signature rs1801280 T341C (I114T) single-nucleotide polymorphism (SNP), whereas NAT2*7B possesses a signature rs1799931 G857A (G286E) SNP. NAT2 alleles possessing the T341C (I114T) or G857A (G286E) SNP were recombinant expressed in yeast and tested for capacity to catalyze the O-acetylation of the N-hydroxy metabolites of heterocyclic amines (HCAs). The T341C (I114T) SNP reduced the O-acetylation of N-hydroxy-2-amino-3-methylimidazo [4,5-f] quinoline (N-OH-IQ), N-hydroxy-2-amino-3,8-dimethylimidazo [4,5-f] quinoxaline (N-OH-MeIQx) and N-hydroxy- 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (N-OH-PhIP), whereas the G857A (G286E) SNP reduced the O-acetylation of N-OH-IQ and N-OH-MeIQx but not N-OH-PhIP. The G857A (G286E) SNP significantly (p < 0.05) reduced apparent Km toward N-OH-PhIP but did not significantly (p > 0.05) affect apparent Vmax. Cultures of DNA repair-deficient Chinese hamster ovary (CHO) cells transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B alleles were incubated with various concentrations of IQ, MeIQx or PhIP and double-stranded DNA damage and reactive oxygen species (ROS) were measured. Transfection with human CYP1A2 did not significantly (p > 0.05) increase HCA-induced DNA damage and ROS over un-transfected cells. Additional transfection with NAT2*4, NAT2*5B or NAT2*7B allele increased both DNA damage and ROS. The magnitude of the increases was both NAT2 allele- and substrate-dependent showing the same pattern as observed for the O-acetylation of the N-hydroxylated HCAs suggesting that both are mediated via NAT2-catalyzed O-acetylation. The results document the role of NAT2 and its genetic polymorphism on the O-acetylation and genotoxicity of HCAs.

Effect of N-acetyltransferase 2 genetic polymorphism on 4,4'-methylenebis(2-chloroaniline)-induced genotoxicity and oxidative stress.

4,4'-Methylenebis(2-chloroaniline) or MOCA is an aromatic amine used primarily in polyurethane and rubber industry. MOCA has been linked to hepatomas in animal studies while limited epidemiologic studies reported the association of exposure to MOCA and urinary bladder and breast cancer. We investigated MOCA-induced genotoxicity and oxidative stress in DNA repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human metabolizing enzymes CYP1A2 and N-acetyltransferase 2 (NAT2) variants as well as in rapid, intermediate, and slow NAT2 acetylator cryopreserved human hepatocytes. N-acetylation of MOCA was highest in UV5/1A2/NAT2*4 followed by UV5/1A2/NAT2*7B and UV5/1A2/NAT2*5B CHO cells. Human hepatocytes showed a NAT2 genotype-dependent response with highest N-acetylation in rapid acetylators followed by intermediate and slow acetylators. MOCA induced higher levels of mutagenesis and DNA damage in UV5/1A2/NAT2*7B compared to UV5/1A2/NAT2*4 and UV5/1A2/NAT2*5B cells (p < 0.0001). MOCA also induced higher levels of oxidative stress in UV5/1A2/NAT2*7B cells. MOCA caused concentration-dependent increase in DNA damage in cryopreserved human hepatocytes (linear trend p < 0.001) which was NAT2 genotype dependent i.e., highest in rapid acetylators, lower in intermediate acetylators, and lowest in slow acetylators (p < 0.0001). Our findings show that N-acetylation and genotoxicity of MOCA is NAT2 genotype dependent and suggest that individuals possessing NAT2*7B are at higher risk to MOCA-induced mutagenicity. DNA damage, and oxidative stress. They confirm significant differences in genotoxicity between the NAT2*5B and NAT2*7B alleles, both of which are associated with slow acetylator phenotype.

Lung cancer-kidney cross talk induces kidney injury, interstitial fibrosis, and enhances cisplatin-induced nephrotoxicity.

Patients with cancer represent a unique patient population with increased susceptibility to kidney disease. Drug-induced acute kidney injury (AKI) in patients with cancer is a common problem. Cisplatin is a highly effective treatment used in many solid-organ cancers and causes AKI in 30% of patients, increasing the risk of chronic kidney disease development. Most preclinical cisplatin toxicity studies have been completed in mice without cancer. We believe that the physiology of patients with cancer is not adequately represented in preclinical models, and the objective of this study was to determine how lung cancer will alter the nephrotoxicity of cisplatin. A genetically engineered mouse model and a syngeneic xenograft model of lung cancer were used. Mice were divided into the following four groups: 1) noncancer/vehicle, 2) noncancer/cisplatin, 3) cancer/vehicle, and 4) cancer/cisplatin. Mice were administered cisplatin via intraperitoneal injection once a week for 4 wk. Animals were euthanized 72 h following their final cisplatin injection. Mice with lung cancer had increased renal toxicity, injury, and fibrosis following repeated low doses of cisplatin. In addition, lung cancer alone induced kidney injury and fibrosis in the kidney before cisplatin treatment. In conclusion, this is the first study that we are aware of that assesses the impact of cancer on the kidney in conjunction with the nephrotoxicity of cisplatin. We believe that cancer is providing the first hit to the kidney and the subsequent damage from repeated doses of cisplatin becomes unsurmountable, leading to AKI and progression to chronic kidney disease.NEW & NOTEWORTHY Patients with cancer have impaired kidney function and increased susceptibility to nephrotoxic agents. Cisplatin is a commonly used chemotherapeutic with nephrotoxicity as the dose-limiting side effect. Cisplatin nephrotoxicity is almost exclusively studied in mice without cancer. Our current preclinical models do not adequately represent the complexity of patients with cancer. This study demonstrates increased renal toxicity, injury, and fibrosis in mice with lung cancer, which is exacerbated with cisplatin treatment. These results highlight the necessity of using preclinical models that more accurately capture the altered physiology of patients with cancer treated with cisplatin.

Upregulation of cytidine deaminase in NAT1 knockout breast cancer cells.

PURPOSE: Arylamine N-acetyltransferase 1 (NAT1), a phase II metabolic enzyme, is frequently upregulated in breast cancer. Inhibition or depletion of NAT1 leads to growth retardation in breast cancer cells in vitro and in vivo. A previous metabolomics study of MDA-MB-231 breast cancer cells suggests that NAT1 deletion leads to a defect in de novo pyrimidine biosynthesis. In the present study, we observed that NAT1 deletion results in upregulation of cytidine deaminase (CDA), which is involved in the pyrimidine salvage pathway, in multiple breast cancer cell lines (MDA-MB-231, MCF-7 and ZR-75-1). We hypothesized that NAT1 KO MDA-MB-231 cells show differential sensitivity to drugs that either inhibit cellular pyrimidine homeostasis or are metabolized by CDA. METHODS: The cells were treated with (1) inhibitors of dihydroorotate dehydrogenase or CDA (e.g., teriflunomide and tetrahydrouridine); (2) pyrimidine/nucleoside analogs (e.g., gemcitabine and 5-azacytidine); and (3) naturally occurring, modified cytidines (e.g., 5-formyl-2'-deoxycytidine; 5fdC). RESULTS: Although NAT1 KO cells failed to show differential sensitivity to nucleoside analogs that are metabolized by CDA, they were markedly more sensitive to 5fdC which induces DNA damage in the presence of high CDA activity. Co-treatment with 5fdC and a CDA inhibitor, tetrahydrouridine, abrogated the increase in 5fdC cytotoxicity in NAT1 KO cells, suggesting that the increased sensitivity of NAT1 KO cells to 5fdC is dependent on their increased CDA activity. CONCLUSIONS: The present findings suggest a novel therapeutic strategy to treat breast cancer with elevated NAT1 expression. For instance, NAT1 inhibition may be combined with cytotoxic nucleosides (e.g., 5fdC) for breast cancer treatment.

N-acetyltransferase 2 genetic polymorphism modifies genotoxic and oxidative damage from new psychoactive substances.

The use of new psychoactive substances (NPS) as drugs of abuse is common and increasingly popular, particularly among youth and neglected communities. Recent studies have reported acute toxic effects from these chemicals; however, their long-term toxicity is unknown. Genetic differences between individuals likely affect the toxicity risk. Arylamine N-acetyltransferase 2 (NAT2) capacity differs among individuals due to genetic inheritance. The goal of the present study is to investigate the gene-environment interaction between NAT2 polymorphism and toxicity after exposure to these chemicals. We measured N-acetylation by human NAT1 and NAT2 and found that N-acetylation of NPS is carried out exclusively by NAT2. Differences in N-acetylation between NAT2*4 (reference allele) and NAT2*5B (common variant allele) were highly significant (p < 0.0001). Using DNA repair-deficient genetically engineered Chinese hamster ovary (CHO cells), expressing human CYP1A2 and either NAT2*4 or NAT2*5B, we measured the induction of DNA double-strand breaks ([Formula: see text]H2Ax) following treatment of the CHO cells with increasing concentrations of NPS. The induction of [Formula: see text]H2Ax showed a NAT2 allele-dependent response, higher in the NAT2*4 vs NAT2*5B alleles (p < 0.05). Induction of oxidative stress (ROS/RNS) was evaluated; we observed NAT2 allele-dependent response for all compounds in concentrations as low as 10 [Formula: see text]M, where NAT2*4 showed increased ROS/RNS vs NAT2*5B (p < 0.05). In summary, NPS are N-acetylated by NAT2 at rates higher in cells expressing NAT2*4 than NAT2*5B. Exposure to psychoactive chemicals results in genotoxic and oxidative damage that is modified by the NAT2 genetic polymorphism.

Association between cigarette smoking and ovarian reserve among women seeking fertility care.

INTRODUCTION: This study examined the association of smoking with ovarian reserve in a cross-sectional study of 207 women enrolled in the Louisville Tobacco Smoke Exposure, Genetic Susceptibility, and Infertility (LOUSSI) Study and assessed effect modification by NAT2 acetylator phenotype. METHODS: Information on current smoking status was collected using a structured questionnaire and confirmed by cotinine assay. Serum anti-Müllerian hormone (AMH) levels were used to assess ovarian reserve. Diminished ovarian reserve (DOR) was defined as AMH <1ng/mL. Single nucleotide polymorphisms in the NAT2 gene, which metabolizes toxins found in cigarette smoke, were analyzed to determine NAT2 acetylator status. Linear and logistic regression were used to determine the effects of smoking on ovarian reserve and evaluate effect modification by NAT2. Regression analyses were stratified by polycystic ovary syndrome (PCOS) status and adjusted for age. RESULTS: Current smoking status, either passive or active as measured by urinary cotinine assay, was not significantly associated with DOR. For dose-response assessed using self-report, the odds of DOR increased significantly for every additional cigarette currently smoked (Odds ratio, OR:1.08; 95% confidence interval, 95%CI:1.01-1.15); additionally, every 1 pack-year increase in lifetime exposure was associated with an increased odds of DOR among women without PCOS (OR: 1.08 95%CI: 0.99-1.18). These trends appear to be driven by the heavy or long-term smokers. Effect modification by NAT2 genotype was not established. CONCLUSION: A history of heavy smoking may indicate increased risk of diminished ovarian reserve.

Acetyl coenzyme A kinetic studies on N-acetylation of environmental carcinogens by human N-acetyltransferase 1 and its NAT1*14B variant.

N-acetyltransferase 1 (NAT1) is a xenobiotic metabolizing enzyme that uses acetyl coenzyme A (AcCoA) as a cofactor for N-acetylation of many carcinogens including aromatic amines and alkylanilines. NAT1 is characterized by single nucleotide polymorphisms (SNPs) that may modulate affinity towards AcCoA. In the current study, we used Chinese hamster ovary (CHO) cells stably transfected with human NAT1*4 (reference allele) or NAT1*14B (variant allele) to measure AcCoA kinetic parameters for N-acetyltransferase activity measurements towards p-aminobenzoic acid (PABA), 4-aminobiphenyl (4-ABP), ß-naphthylamine (BNA), benzidine and 3,4-dimethylaniline (3,4-DMA). Our results showed higher N-acetylation rates for each substrate catalyzed by NAT1*4 compared to NAT1*14B. NAT1*4 exhibited higher affinity to AcCoA when catalyzing the N-acetylation of BNA and benzidine compared to NAT1*14B. The results of the current study provide further insights into differences in carcinogen metabolism among individuals possessing the NAT1*14B haplotype.

Dataset for proteomic analysis of arylamine N-acetyltransferase 1 knockout MDA-MB-231 breast cancer cells.

Arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer. An unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines were performed. Among 4,890 proteins identified, 737 and 651 proteins were found significantly (p < 0.01) upregulated and downregulated, respectively, in NAT1 KO cells, compared to the parental cells. Each set of proteins was analyzed to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in the set. Among the proteins upregulated in NAT1 KO cells, processes associated with MHC major histocompatibility complex I-mediated antigen presentation were significantly enriched. Multiple processes involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells. The current dataset contains additional representations of the biological processes and components that are differentially enriched in NAT1 KO MDA-MB-231 cells and will serve as a basis for generating novel hypotheses regarding the role of NAT1 in breast cancer. Data are available via ProteomeXchange with identifier PXD035953.

F4/80hi Resident Macrophages Contribute to Cisplatin-Induced Renal Fibrosis.

Background: Cisplatin-induced kidney injury remains a major obstacle in utilizing cisplatin as a chemotherapeutic for solid-organ cancers. Thirty percent of patients treated with cisplatin develop acute kidney injury (AKI), and even patients who do not develop AKI are at risk for long-term declines in kidney function and development of chronic kidney disease (CKD). Modeling cisplatin-induced kidney injury in mice has revealed that repeated low doses of cisplatin lead to development of kidney fibrosis. This model can be used to examine AKI-to-CKD transition processes. Macrophages play a role in some of these processes, including immune response, wound healing, and tissue remodeling. Depleting macrophage populations in the kidney reduced fibrosis development in other models of renal fibrosis. Methods: We used either C57BL/6 mice with a Ccr2 genetic knockout or liposome encapsulated clodronate (Clodrosome) to deplete macrophage populations during repeated 9 mg/kg cisplatin treatments. We assessed how immune cell populations were altered in the blood and kidney of these mice and how these alterations affected development of renal fibrosis and kidney injury. Results: We found that Clodrosome treatment decreased collagen deposition, myofibroblast accumulation, and inflammatory cytokine production, whereas Ccr2 genetic knockout had no effect on these markers after cisplatin treatment. Additionally, Ccr2-/- mice had decreased levels of F4/80lo infiltrating macrophages in the kidney after cisplatin treatments, but Clodrosome treatment depleted F4/80hi resident and CD206+ M2 macrophages. Conclusions: These data suggest that Clodrosome depletion of F4/80hi and M2 macrophages in the kidney attenuates development of renal fibrosis after repeated low doses of cisplatin.

N-acetyltransferase 2 acetylator genotype-dependent N-acetylation and toxicity of the arylamine carcinogen ß-naphthylamine in cryopreserved human hepatocytes.

We used cryopreserved human hepatocytes that express rapid, intermediate, and slow acetylator N-acetyltransferase 2 (NAT2) genotypes to measure the N-acetylation of ß-naphthylamine (BNA) which is one of the aromatic amines found in cigarette smoke including E-cigarettes. We investigated the role of NAT2 genetic polymorphism in genotoxicity and oxidative stress induced by BNA. In vitro BNA NAT2 activities in rapid acetylators was 1.6 and 3.5-fold higher than intermediate (p < 0.01) and slow acetylators (p < 0.0001). BNA N-acetylation in situ was 3 to 4- fold higher in rapid acetylators than slow acetylators, following incubation with 10 and 100 µM BNA (p < 0.01). DNA damage was two to threefold higher in the rapid versus slow acetylators (p < 0.0001) and 2.5-fold higher in intermediate versus slow acetylators following BNA treatment at 100 and 1000 µM, ROS/RNS level was the highest in rapid acetylators followed by intermediate and then slow acetylators (p < 0.0001). Our findings show that the N-acetylation of BNA is NAT2 genotype dependent in cryopreserved human hepatocytes and our data further document an important role for NAT2 genetic polymorphism in modifying BNA-induced genotoxicity and oxidative damage.

Proteomic analysis of arylamine N-acetyltransferase 1 knockout breast cancer cells: Implications in immune evasion and mitochondrial biogenesis.

Previous studies have shown that inhibition or depletion of N-acetyltransferase 1 (NAT1) in breast cancer cell lines leads to growth retardation both in vitro and in vivo, suggesting that NAT1 contributes to rapid growth of breast cancer cells. To understand molecular and cellular processes that NAT1 contributes to and generate novel hypotheses in regard to NAT1's role in breast cancer, we performed an unbiased analysis of proteomes of parental MDA-MB-231 breast cancer cells and two separate NAT1 knockout (KO) cell lines. Among 4890 proteins identified, 737 proteins were found significantly (p < 0.01) upregulated, and 651 proteins were significantly (p < 0.01) downregulated in both NAT1 KO cell lines. We performed enrichment analyses to identify Gene Ontology biological processes, molecular functions, and cellular components that were enriched in each data set. Among the proteins upregulated in NAT1 KO cells, pathways associated with MHC (major histocompatibility complex) I-mediated antigen presentation were significantly enriched. This raises an interesting and new hypothesis that upregulation of NAT1 in breast cancer cells may aid them evade immune detection. Multiple pathways involved in mitochondrial functions were collectively downregulated in NAT1 KO cells, including multiple subunits of mitochondrial ATP synthase (Complex V of the electron transport chain). This was accompanied by a reduction in cell cycle-associated proteins and an increase in pro-apoptotic pathways in NAT1 KO cells, consistent with reported observations that NAT1 KO cells exhibit a slower growth rate both in vitro and in vivo. Thus, mitochondrial dysfunction in NAT1 KO cells likely contributes to growth retardation.

Differences in ß-naphthylamine metabolism and toxicity in Chinese hamster ovary cell lines transfected with human CYP1A2 and NAT2*4, NAT2*5B or NAT2*7B N-acetyltransferase 2 haplotypes.

ß-naphthylamine (BNA) is an important aromatic amine carcinogen. Current exposures derive primarily from cigarette smoking including e-cigarettes. Occupational and environmental exposure to BNA is associated with urinary bladder cancer which is the fourth most frequent cancer in the United States. N-acetyltransferase 2 (NAT2) is an important metabolizing enzyme for aromatic amines. Previous studies investigated mutagenicity and genotoxicity of BNA in bacteria and in rabbit or rat hepatocytes. However, the effects of human NAT2 genetic polymorphism on N-acetylation and genotoxicity induced by BNA still need to be clarified. We used nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells that were stably transfected with human CYP1A2 and NAT2 alleles: NAT2*4 (reference allele), NAT2*5B (variant slow acetylator allele common in Europe) or NAT2*7B (variant slow acetylator allele common in Asia). BNA N-acetylation was measured both in vitro and in situ via high-performance liquid chromatography (HPLC). Hypoxanthine phosphoribosyl transferase (HPRT) mutations, double-strand DNA breaks, and reactive oxygen species (ROS) were measured as indices of toxicity. NAT2*4 cells showed significantly higher BNA N-acetylation rates followed by NAT2*7B and NAT2*5B. BNA caused concentration-dependent increases in DNA damage and ROS levels. NAT2*7B showed significantly higher levels of HPRT mutants, DNA damage and ROS than NAT2*5B (p < 0.001, p < 0.0001, p < 0.0001 respectively) although both are slow alleles. Our findings suggest that BNA N-acetylation and toxicity are modified by NAT2 polymorphism. Furthermore, they confirm heterogeneity among slow acetylator alleles for BNA metabolism and toxicity supporting differential risk for individuals carrying NAT2*7B allele.

Pharmacological inhibitors of autophagy have opposite effects in acute and chronic cisplatin-induced kidney injury.

The nephrotoxicity of cisplatin remains a major hurdle in the field of oncology. Thirty percent of patients treated with cisplatin develop acute kidney injury, and all patients are at risk for long-term impacts on kidney function. There are currently no Federal Drug Administration-approved agents to prevent or treat cisplatin-induced kidney injury. The dosing regimen used in preclinical models of nephrotoxicity may impact the success of therapeutic candidates in clinical trials. Here, we demonstrated that pharmacological inhibitors of autophagy have opposite effects when used as interventions in two different models of cisplatin-induced kidney injury. Eight-week-old male C57BL/6 mice were treated with either one dose of 20 mg/kg cisplatin or weekly doses of 9 mg/kg cisplatin for 4 wk or until body weight loss exceeded 30%. Concurrently, mice were administered multiple doses of 60 mg/kg chloroquine or 15 mg/kg 3-methyladenine attempting to globally inhibit autophagy. Mice that received a single high dose of cisplatin had worsened kidney function, inflammation, and cell death with the addition of chloroquine. 3-Methlyadenine did not impact the development of acute kidney injury in this model. In contrast, mice that received repeated low doses of cisplatin showed improved kidney function, reduced inflammation, and reduced fibrosis when treated with either chloroquine or 3-methyladenine. This study highlights how therapeutic candidates can have drastically different effects on the development of cisplatin-induced kidney injury depending on the dosing model used. This emphasizes the importance of choosing the appropriate model of injury for preclinical studies.NEW & NOTEWORTHY This study examined how inhibition of autophagy has opposite effects on the development of acute and chronic kidney injury. Autophagy inhibition exacerbated the development of acute kidney injury following a single high dose of cisplatin but prevented the development of injury and fibrosis following repeated low doses of cisplatin.

Hexavalent chromium increases the metabolism and genotoxicity of aromatic amine carcinogens 4-aminobiphenyl and ß-naphthylamine in immortalized human lung epithelial cells.

Humans are exposed to carcinogenic chemicals via occupational and environmental exposures. Common chemicals of concern that can occur in exposures together are aromatic amines (e.g., 4-aminobiphenyl [4-ABP] and ß-naphthylamine [BNA]) and hexavalent chromium (Cr[VI]). Arylamine N-acetyltransferases 1 and 2 (NAT1 and NAT2) are key to the metabolism of aromatic amines and their genotoxicity. The effects of Cr(VI) on the metabolism of aromatic amines remains unknown as well as how it may affect their ensuing toxicity. The objective of the research presented here is to investigate the effects of Cr(VI) on the metabolism and genotoxicity of 4-ABP and BNA in immortalized human lung epithelial cells (BEP2D) expressing NAT1 and NAT2. Exposure to Cr(VI) for 48 h increased NAT1 activity (linear regression analysis: P < 0.0001) as measured by N-acetylation of para-aminobenzoic acid (PABA) in BEP2D cells but not NAT2 N-acetylation of sulfamethazine, which are prototypic NAT1 and NAT2 substrates respectively. Cr(VI) also increased the N-acetylation of 4-ABP and BNA. In BEP2D cells the N-acetylation of 4-ABP (1-3 µM) exhibited a dose-dependent increase (linear regression analysis: P < 0.05) following co-incubation with 0-3 µM Cr(VI). In BEP2D cells, incubation with Cr(VI) caused dose-dependent increases (linear regression analysis: P < 0.01) in expression of CYP1A1 protein and catalytic activity. For genotoxicity, BEP2D cells were exposed to 4-ABP or BNA with/without Cr(VI) for 48 h. We observed dose-dependent increases (linear regression analysis: P < 0.01) in phospho-γH2AX protein expression for combined treatment of 4-ABP or BNA with Cr(VI). Further using a CYP1A1 inhibitor (α-naphthoflavone) and NAT1 siRNA, we found that CYP1A1 inhibition did not reduce the increased N-acetylation or genotoxicity of BNA by Cr(VI), while NAT1 inhibition did reduce increases in BNA N-acetylation and genotoxicity by Cr(VI). We conclude that during co-exposure of aromatic amines and Cr(VI) in human lung cells, Cr(VI) increased NAT1 activity contributing to increased 4-ABP and BNA genotoxicity.

560G>A (rs4986782) (R187Q) Single Nucleotide Polymorphism in Arylamine N-Acetyltransferase 1 Increases Affinity for the Aromatic Amine Carcinogens 4-Aminobiphenyl and N-Hydroxy-4-Aminobiphenyl: Implications for Cancer Risk Assessment.

Human arylamine N-acetyltransferase 1 (NAT1) catalyzes the N-acetylation of arylamine carcinogens such as 4-aminobiphenyl (ABP), and following N-hydroxylation, the O-acetylation of N-hydroxy-arylamine carcinogens such as N-hydroxy-ABP (N-OH-ABP). Genetic polymorphisms in NAT1 are linked to cancer susceptibility following exposures. The effects of individual single nucleotide polymorphisms (SNPs) in the NAT1 coding exon on Michaelis-Menten kinetic constants was assessed for ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activity following transfection of human NAT1 into COS-1 cells (SV40-transformed African green monkey kidney cells). NAT1 coding region SNPs 97C > T (rs56318881) (R33stop), 190C > T (rs56379106) (R64W), 559C > T (rs5030839) (R187stop) and 752A > T (rs56172717) (D251V) reduced ABP N- acetyltransferase and N-OH-ABP O-acetyltransferase activity below detection. 21T > G (rs4986992) (synonymous), 402T > C (rs146727732) (synonymous), 445G > A (rs4987076) (V149I), 613A > G (rs72554609) (M205V) and 640T > G (rs4986783) (S241A) did not significantly affect Vmax for ABP N-acetyltransferase or N-OH-ABP O-acetyltransferase. 781G > A (rs72554610) (E261K), and 787A > G (rs72554611) (I263V) slightly reduced ABP N-acetyltransferase and N-OH-ABP O-acetyltransferase activities whereas 560G > A (rs4986782) (R187Q) substantially and significantly reduced them. 560G > A (rs4986782) (R187Q) significantly reduced the apparent Km for ABP and N-OH-ABP a finding that was not observed with any of the other NAT1 SNPs tested. These findings suggest that the role of the 560G > A (rs4986782) (R187Q) SNP cancer risk assessment may be modified by exposure level to aromatic amine carcinogens such as ABP.

Human N-Acetyltransferase 1 and 2 Differ in Affinity Towards Acetyl-Coenzyme A Cofactor and N-Hydroxy-Arylamine Carcinogens.

Arylamine N-acetyltransferases catalyze the transfer of acetyl groups from the endogenous cofactor acetyl coenzyme A (AcCoA) to arylamine (N-acetylation) and N-hydroxy-arylamine (O-acetylation) acceptors. Humans express two arylamine N-acetyltransferase isozymes (NAT1 and NAT2) which catalyze both N- and O-acetylation but differ in genetic regulation, substrate selectivity, and expression in human tissues. We investigated recombinant human NAT1 and NAT2 expressed in an Escherichia coli JM105 and Schizosaccharomyces pombe expression systems as well as in Chinese hamster ovary (CHO) cells to assess the relative affinity of AcCoA for human NAT1 and NAT2. NAT1 and NAT2 affinity for AcCoA was higher for recombinant human NAT1 than NAT2 when catalyzing N-acetylation of aromatic amine carcinogens 2-aminofluroene (AF), 4-aminobiphenyl (ABP), and ß-naphthylamine (BNA) and the metabolic activation of N-hydroxy-2-aminofluorene (N-OH-AF) and N-hydroxy-4-aminobiphenyl (N-OH-ABP) via O-acetylation. These results suggest that AcCoA level may influence differential rates of arylamine carcinogen metabolism catalyzed by NAT1 and NAT2 in human tissues. Affinity was higher for NAT2 than for NAT1 using N-OH-AF and N-OH-ABP as substrate consistent with a larger active site for NAT2. In conclusion, following recombinant expression in bacteria, yeast, and CHO cells, we report significant differences in affinity between human NAT1 and NAT2 for its required co-factor AcCoA, as well as for N-hydroxy-arylamines activated via O-acetylation. The findings provide important information to understand the relative contribution of human NAT1 vs NAT2 towards N-acetylation and O-acetylation reactions in human hepatic and extrahepatic tissues.

Expression of arylamine N-acetyltransferase 2 activity in immortalized human bronchial epithelial cells.

Lung cancer is the leading cause of cancer deaths in the United States with high incidence in tobacco smokers. Arylamine N-acetyltransferase 2 (NAT2) is a xenobiotic enzyme that catalyzes both N- and O-acetylation of carcinogens present in tobacco smoke and contributes towards the genotoxicity of these carcinogens. NAT2 allelic variants result in slow, intermediate, and rapid acetylation phenotypes. A recent meta-analysis reported NAT2 non-rapid (slow and intermediate) phenotypes had a significantly increased risk of lung cancer. NAT2 activity in humans is thought to be restricted to liver and gastrointestinal tract, and no studies to our knowledge have reported the expression of NAT2 activity in immortalized human lung epithelial cells. Given the importance of NAT2 in cancer and inhalation of various carcinogens directly into the lungs, we investigated NAT2 activity in human lung epithelial cells. Both NAT1 and NAT2 protein were detected by "in-cell" Western. Arylamine N-acetyltransferase activity was determined with selective substrates for NAT1 (p-aminobenzoic acid; PABA) and NAT2 (sulfamethazine; SMZ) in the presence and absence of a selective NAT1 inhibitor. PABA N-acetylation (NAT1 activity) in cell protein lysates was abolished in the presence of 25 µM of NAT1 inhibitor whereas SMZ N-acetylation (NAT2) was unaffected. Incubation with the NAT1 inhibitor partially reduced the N-acetylation of ß-naphthylamine and the O-acetylation of N-hydroxy-4-aminobiphenyl consistent with catalysis by both NAT1 and NAT2. Immortalized human lung epithelial cells exhibited dose-dependent N-acetylation of 4-ABP with an apparent KM of 24.4 ± 5.1 µM. These data establish that NAT2 is expressed and functional in immortalized human lung epithelial cells and will help us further our understanding of NAT2 in lung cancer.

Neutral ceramidase-dependent regulation of macrophage metabolism directs intestinal immune homeostasis and controls enteric infection.

It is not clear how the complex interactions between diet and intestinal immune cells protect the gut from infection. Neutral ceramidase (NcDase) plays a critical role in digesting dietary sphingolipids. We find that NcDase is an essential factor that controls intestinal immune cell dynamics. Mice lacking NcDase have reduced cluster of differentiation (CD) 8αß+ T cells and interferon (IFN)-γ+ T cells and increased macrophages in the intestine and fail to clear bacteria after Citrobacter rodentium infection. Mechanistically, cellular NcDase or extracellular vesicle (EV)-related NcDase generates sphingosine, which promotes macrophage-driven Th1 immunity. Loss of NcDase influences sphingosine-controlled glycolytic metabolism in macrophages, which regulates the bactericidal activity of macrophages. Importantly, administration of dietary sphingomyelin and genetic deletion or pharmacological inhibition of SphK1 can protect against C. rodentium infection. Our findings demonstrate that sphingosine profoundly alters macrophage glycolytic metabolism, leading to intestinal macrophage activation and T cell polarization, which prevent pathogen colonization of the gut.

Arylamine N-Acetyltransferase 1 Activity is Regulated by the Protein Acetylation Status.

Arylamine N-acetyltransferase 1 (NAT1) is a drug metabolizing enzyme that influences cancer cell proliferation and survival, especially in breast cancer. Lysine-acetylation is an important Post-Translational Modification (PTM) in the regulation of diverse cellular processes. Histone deacetylases (HDACs) and Sirtuins (SIRT) may have an important role on the NAT1 acetylation status, affecting its catalytic capacity and having an impact on the downstream functions of this protein. The aim of the present work is to investigate the acetylation status of NAT1 in human breast cancer. Breast cancer cell lines MDA-MB-231 (ER-, PR-, HER2-) and ZR-75-1 (estrogen receptor+, PR+, HER2+) were cultured in the presence of HDAC inhibitors (SAHA, TSA) or Sirtuin inhibitors (AGK2, EX527, Sirtinol). Under these conditions, NAT1 protein and gene expression as well as enzymatic activity were quantified. Acetylation of NAT1 protein was evaluated following an immunoprecipitation protocol and acetyl-Lysine quantification. Sirt1 and Sirt2 knockdown were performed and NAT1 protein and NAT1 mRNA expression and catalytic activity were quantified. The treatment of MDA-MB-231 or ZR-75-1 cells with increasing HDAC inhibitors resulted in 2 to 15-fold upregulation in NAT1 message expression. Finally, the catalytic activity of NAT1 in the presence of HDAC inhibition increased 2-fold. Conversely, the inhibition of Sirtuin activity did not cause significant changes in NAT1 message but produced a significant decrease in NAT1 catalytic activity. NAT1 acetylation was higher in the cells treated with HDAC inhibitors, as well as Sirtuin inhibitors. Finally, silencing of Sirt1 and Sirt2 genes by siRNA transient knockdown of each or both genes resulted in reduction of NAT1 protein expression and catalytic activity. The use of HDAC and Sirtuin inhibitors has been demonstrated as a promising powerful therapeutic alternative in various cancers. These inhibitors can significantly attenuate tumor burden by limiting tumor growth and metastasis. These compounds can also induce DNA damage, cell cycle arrest, apoptosis, and autophagy to promote cancer cell death. Several studies have shown that NAT1 is upregulated in cancer cells. The results of the present study show that the acetylation status of NAT1 is an important factor that might have a relevant role in the progression of cancer.