Assessment of the impact of mitochondrial genotype upon drug-induced mitochondrial dysfunction in platelets derived from healthy volunteers.

Mitochondrial DNA (mtDNA) is highly polymorphic and encodes 13 proteins which are critical to the production of ATP via oxidative phosphorylation. As mtDNA is maternally inherited and undergoes negligible recombination, acquired mutations have subdivided the human population into several discrete haplogroups. Mitochondrial haplogroup has been found to significantly alter mitochondrial function and impact susceptibility to adverse drug reactions. Despite these findings, there are currently limited models to assess the effect of mtDNA variation upon susceptibility to adverse drug reactions. Platelets offer a potential personalised model of this variation, as their anucleate nature offers a source of mtDNA without interference from the nuclear genome. This study, therefore, aimed to determine the effect of mtDNA variation upon mitochondrial function and drug-induced mitochondrial dysfunction in a platelet model. The mtDNA haplogroup of 383 healthy volunteers was determined using next-generation mtDNA sequencing (Illumina MiSeq). Subsequently, 30 of these volunteers from mitochondrial haplogroups H, J, T and U were recalled to donate fresh, whole blood from which platelets were isolated. Platelet mitochondrial function was tested at basal state and upon treatment with compounds associated with both mitochondrial dysfunction and adverse drug reactions, flutamide, 2-hydroxyflutamide and tolcapone (10-250 µM) using extracellular flux analysis. This study has demonstrated that freshly-isolated platelets are a practical, primary cell model, which is amenable to the study of drug-induced mitochondrial dysfunction. Specifically, platelets from donors of haplogroup J have been found to have increased susceptibility to the inhibition of complex I-driven respiration by 2-hydroxyflutamide. At a time when individual susceptibility to adverse drug reactions is not fully understood, this study provides evidence that inter-individual variation in mitochondrial genotype could be a factor in determining sensitivity to mitochondrial toxicants associated with costly adverse drug reactions.

Exome Sequencing Reveals Common and Rare Variants in F5 Associated With ACE Inhibitor and Angiotensin Receptor Blocker-Induced Angioedema.

Angioedema occurring in the head and neck region is a rare and sometimes life-threatening adverse reaction to angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs). Few studies have investigated the association of common variants with this extreme reaction, but none have explored the combined influence of rare variants yet. Adjudicated cases of ACEI-induced angioedema (ACEI-AE) or ARB-induced angioedema (ARB-AE) and controls were recruited at five different centers. Sequencing of 1,066 samples (408 ACEI-AE, ARB-AE, and 658 controls) was performed using exome-enriched sequence data. A common variant of the F5 gene that causes an increase in blood clotting (rs6025, p.Arg506Gln, also called factor V Leiden), was significantly associated with both ACEI-AE and ARB-AE (odds ratio: 2.85, 95% confidence interval (CI), 1.89-4.25). A burden test analysis of five rare missense variants in F5 was also found to be associated with ACEI-AE or ARB-AE, P = 2.09 × 10-3 . A combined gene risk score of these variants, and the common variants rs6025 and rs6020, showed that individuals carrying at least one variant had 2.21 (95% CI, 1.49-3.27, P = 6.30 × 10-9 ) times the odds of having ACEI-AE or ARB-AE. The increased risk due to the common Leiden allele was confirmed in a genome-wide association study from the United States. A high risk of angioedema was also observed for the rs6020 variant that is the main coagulation defect-causing variant in black African and Asian populations. We found that deleterious missense variants in F5 are associated with an increased risk of ACEI-AE or ARB-AE.

The effect of trichloroethylene metabolites on the hepatic vitamin B12-dependent methionine salvage pathway and its relevance to increased excretion of formic acid in the rat.

The industrial solvent trichloroethylene (TCE) and its two major metabolites trichloroethanol (TCE-OH) and trichloroacetic acid (TCA) cause formic aciduria in male F344 rats. Prior treatment of male F344 rats with 1-aminobenzotriazole a cytochrome P450 inhibitor, followed by TCE (16mk/kg, po), completely prevented formic aciduria, but had no effect on formic acid excretion produced by TCA (8 or 16 mg/kg, po), suggesting TCA may be the proximate metabolite producing this response. Dow and Green reported an increase in the concentration of 5-methyltetrahydrofolate (5-MTHF) in the plasma of rats treated with TCE-OH, suggesting a block in the cycling of 5-MTHF to tetrahydrofolate (THF). This pathway is under the control of the vitamin B12-dependent methionine salvage pathway. We therefore treated rats with three daily doses of methylcobalamin (CH3Cbl) or hydroxocobalamin (OHCbl), a cofactor for methionine synthase, or L-methionine, followed by TCE (16 mg/kg) to determine if they could alleviate the formic aciduria. These pretreatments only partially reduced the excretion of formic acid in the urine. Although prior treatment with S-adenosyl-L-methionine had no effect on formic acid excretion. Consistent with these findings, the activity of methionine synthase in the liver of TCE-treated rats was not inhibited. Transcriptomic analysis of the liver-identified nine differential expressed genes, of note, was downregulation of Lmbrd1 involved in the conversion of vitamin B12 into CH3Cbl, a cofactor for methionine synthase. Our findings indicate that the formic aciduria produced by TCE-OH and TCA may be the result of a block in the recycling of 5-MTHF to THF, the effect on the methionine salvage pathway being a secondary response following acute exposure.

Pharmacogenomics of statin-related myopathy: Meta-analysis of rare variants from whole-exome sequencing.

AIMS: Statin-related myopathy (SRM), which includes rhabdomyolysis, is an uncommon but important adverse drug reaction because the number of people prescribed statins world-wide is large. Previous association studies of common genetic variants have had limited success in identifying a genetic basis for this adverse drug reaction. We conducted a multi-site whole-exome sequencing study to investigate whether rare coding variants confer an increased risk of SRM. METHODS AND RESULTS: SRM 3-5 cases (N = 505) and statin treatment-tolerant controls (N = 2047) were recruited from multiple sites in North America and Europe. SRM 3-5 was defined as symptoms consistent with muscle injury and an elevated creatine phosphokinase level >4 times upper limit of normal without another likely cause of muscle injury. Whole-exome sequencing and variant calling was coordinated from two analysis centres, and results of single-variant and gene-based burden tests were meta-analysed. No genome-wide significant associations were identified. Given the large number of cases, we had 80% power to identify a variant with minor allele frequency of 0.01 that increases the risk of SRM 6-fold at genome-wide significance. CONCLUSIONS: In this large whole-exome sequencing study of severe statin-related muscle injury conducted to date, we did not find evidence that rare coding variants are responsible for this adverse drug reaction. Larger sample sizes would be required to identify rare variants with small effects, but it is unclear whether such findings would be clinically actionable.

Susceptibility to corticosteroid-induced adrenal suppression: a genome-wide association study.

BACKGROUND: A serious adverse effect of corticosteroid therapy is adrenal suppression. Our aim was to identify genetic variants affecting susceptibility to corticosteroid-induced adrenal suppression. METHODS: We enrolled children with asthma who used inhaled corticosteroids as part of their treatment from 25 sites across the UK (discovery cohort), as part of the Pharmacogenetics of Adrenal Suppression with Inhaled Steroids (PASS) study. We included two validation cohorts, one comprising children with asthma (PASS study) and the other consisting of adults with chronic obstructive pulmonary disorder (COPD) who were recruited from two UK centres for the Pharmacogenomics of Adrenal Suppression in COPD (PASIC) study. Participants underwent a low-dose short synacthen test. Adrenal suppression was defined as peak cortisol less than 350 nmol/L (in children) and less than 500 nmol/L (in adults). A case-control genome-wide association study was done with the control subset augmented by Wellcome Trust Case Control Consortium 2 (WTCCC2) participants. Single nucleotide polymorphisms (SNPs) that fulfilled criteria to be advanced to replication were tested by a random-effects inverse variance meta-analysis. This report presents the primary analysis. The PASS study is registered in the European Genome-phenome Archive (EGA). The PASS study is complete whereas the PASIC study is ongoing. FINDINGS: Between November, 2008, and September, 2011, 499 children were enrolled to the discovery cohort. Between October, 2011, and December, 2012, 81 children were enrolled to the paediatric validation cohort, and from February, 2010, to June, 2015, 78 adults were enrolled to the adult validation cohort. Adrenal suppression was present in 35 (7%) children in the discovery cohort and six (7%) children and 17 (22%) adults in the validation cohorts. In the discovery cohort, 40 SNPs were found to be associated with adrenal suppression (genome-wide significance p<1 × 10-6), including an intronic SNP within the PDGFD gene locus (rs591118; odds ratio [OR] 7·32, 95% CI 3·15-16·99; p=5·8 × 10-8). This finding for rs591118 was validated successfully in both the paediatric asthma (OR 3·86, 95% CI 1·19-12·50; p=0·02) and adult COPD (2·41, 1·10-5·28; p=0·03) cohorts. The proportions of patients with adrenal suppression by rs591118 genotype were six (3%) of 214 patients with the GG genotype, 15 (6%) of 244 with the AG genotype, and 22 (25%) of 87 with the AA genotype. Meta-analysis of the paediatric cohorts (discovery and validation) and all three cohorts showed genome-wide significance of rs591118 (respectively, OR 5·89, 95% CI 2·97-11·68; p=4·3 × 10-9; and 4·05, 2·00-8·21; p=3·5 × 10-10). INTERPRETATION: Our findings suggest that genetic variation in the PDGFD gene locus increases the risk of adrenal suppression in children and adults who use corticosteroids to treat asthma and COPD, respectively. FUNDING: Department of Health Chair in Pharmacogenetics.

Aristolochic acids - Induced transcriptomic responses in rat renal proximal tubule cells in vitro.

Aristolochic acids (AAs) are the active components of herbal drugs derived from Aristolochia species that have been used for medicinal purposes since antiquity. However, AAs have recently been discovered to be highly nephrotoxic and induced urothelial cancer in humans and malignant tumors in the kidney and urinary tract of rodents. In this study, we exposed rat renal proximal tubule cells in vitro to a sub-cytotoxic level of AAs at three different time points (6 h, 24 h and 72 h). We then analyzed the gene expression profile after the compound exposure. Functional analysis with Ingenuity Pathways Analysis and DAVID tools revealed that at the late time point (72 h) there are many significantly altered genes involved in cancer-related pathways such as p53 signaling. MIAMI-compliant microarray data are deposited in the NCBI GEO database under accession number GSE68687 and can be found at: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE68687.

Pharmacogenetics of antiepileptic drug-induced hypersensitivity.

Antiepileptic drugs can induce potentially life-threatening hypersensitivity reactions such as Stevens-Johnson syndrome at a frequency of one in 10,000 to one in 1000 treated patients. There is a considerable cross-reactivity among different antiepileptic drugs but the mechanisms are not known. In this review we have summarized current evidence on antiepileptic drug-induced hypersensitivity reactions and performed meta-analyses of published case-control studies that investigated associations between HLA alleles and several antiepileptic drugs in diverse populations. As the heterogeneity between studies was high, we conducted subsequent subgroup analyses and showed that HLA-B*15:02 was associated with carbamazepine, lamotrigine and phenytoin-induced Stevens-Johnson syndrome in Asian populations indicating that pretreatment testing may prevent cross-reactivity. Additionally, we explored the potential of new, high-throughput technologies that may help to understand the mechanisms and predict the risk of adverse drug reactions in the future.

Oxidative stress induced by potassium bromate exposure results in altered tight junction protein expression in renal proximal tubule cells.

Potassium bromate (KBrO(3)) is an oxidising agent that has been widely used in the food and cosmetic industries. It has shown to be both a nephrotoxin and a renal carcinogen in in vivo and in vitro models. Here, we investigated the effects of KBrO(3) in the human and rat proximal tubular cell lines RPTEC/TERT1 and NRK-52E. A genome-wide transcriptomic screen was carried out from cells exposed to a sub-lethal concentration of KBrO(3) for 6, 24 and 72 h. Pathway analysis identified "glutathione metabolism", "Nrf2-mediated oxidative stress" and "tight junction (TJ) signalling" as the most enriched pathways. TJ signalling was less impacted in the rat model, and further studies revealed low transepithelial electrical resistance (TEER) and an absence of several TJ proteins in NRK-52E cells. In RPTEC/TERT1 cells, KBrO(3) exposure caused a decrease in TEER and resulted in altered expression of several TJ proteins. N-Acetylcysteine co-incubation prevented these effects. These results demonstrate that oxidative stress has, in conjunction with the activation of the cytoprotective Nrf2 pathway, a dramatic effect on the expression of tight junction proteins. The further understanding of the cross-talk between these two pathways could have major implications for epithelial repair, carcinogenesis and metastasis.

Transcriptomic alterations induced by Ochratoxin A in rat and human renal proximal tubular in vitro models and comparison to a rat in vivo model.

Ochratoxin A (OTA) is a widely studied compound due to its role in renal toxicity and carcinogenicity. However, there is still no consensus on the exact mechanisms of toxicity or carcinogenicity. In the current study, we analysed the effect of OTA on three human renal proximal tubular models (human primary, RPTEC/TERT1 and HK-2 cells) and two rat renal proximal tubular models (rat primary and NRK-52E cells). Global transcriptomics analysis at two exposure times was performed to generate a set of 756 OTA sensitive genes. This gene set was then compared in more detail across all models and additionally to a rat in vivo renal cortex model. The results demonstrate a well-conserved response across all models. OTA resulted in deregulation of a number of pathways including cytoskeleton, nucleosome regulation, translation, transcription, ubiquitination and cell cycle pathways. Interestingly, the oxidative stress activated Nrf2 pathway was not enriched. These results point to an epigenetic action of OTA, perhaps initiated by actin binding as the actin remodelling gene, advillin was the highest up-regulated in all models. The largest model differences were observed between the human and the rat in vitro models. However, since the human in vitro models were more similar to the rat in vivo model, it is more likely that these differences are model-specific rather than species-specific per se. This study demonstrates the usefulness of in vitro cell culture models combined with transcriptomic analysis for the investigation of mechanisms of toxicity and carcinogenicity. In addition, these results provide further evidence supporting a non-genotoxic mechanism of OTA-induced carcinogenicity.