The clastogenicity of 4NQO is cell-type dependent and linked to cytotoxicity, length of exposure and p53 proficiency.

4-Nitroquinoline 1-oxide (4NQO) is used as a positive control in various genotoxicity assays because of its known mutagenic and carcinogenic properties. The chemical is converted into 4-hydroxyaminoquinoline 1-oxide and gives rise to three main DNA adducts, N-(deoxyguanosin-8-yl)-4AQO, 3-(desoxyguanosin-N (2)-yl)-4AQO and 3-(deoxyadenosin-N (6)-yl)-4AQO. This study was designed to assess the shape of the dose-response curve at low concentrations of 4NQO in three human lymphoblastoid cell lines, MCL-5, AHH-1 and TK6 as well as the mouse lymphoma L5178Y cell line in vitro. Chromosomal damage was investigated using the in vitro micronucleus assay, while further gene mutation and DNA damage studies were carried out using the hypoxanthine-guanine phosphoribosyltransferase forward mutation and comet assays. 4NQO showed little to no significant increases in micronucleus induction in the human lymphoblastoid cell lines, even up to 55±5% toxicity. A dose-response relationship could only be observed in the mouse lymphoma cell line L5178Y after 4NQO treatment, even at concentrations with no reduction in cell viability. Further significant increases in gene mutation and DNA damage induction were observed. Hence, 4NQO is a more effective point mutagen than clastogen, and its suitability as a positive control for genotoxicity testing has to be evaluated for every individual assay.

Metabolic syndrome is associated with prostate enlargement: a systematic review, meta-analysis, and meta-regression on patients with lower urinary tract symptom factors.

BACKGROUND: Metabolic syndrome (MetS) is defined by at least three of the following five criteria: blood pressure ⩾130/85 mmHg, fasting blood glucose ⩾5.6 mmol/l, triglycerides concentration ⩾1.7 mmol/l, waist circumference ⩾102 cm (for men), and high-density lipoprotein cholesterol concentration <1.03 mmol/l (for men). MetS has been associated with worse lower urinary tract symptoms (LUTS) and higher International Prostate Symptom questionnaire scores. MATERIALS AND METHODS: MEDLINE, Cochrane, ClinicalTrials.gov, and SCOPUS were critically appraised for all peer-reviewed manuscripts that suitably fulfilled our protocol's inclusion criteria established a priori. Meta-analytical and meta-regression calculations were performed in R using the Sidik-Jonkman and Hartung-Knapp random effects model and predefined covariates. RESULTS: A total of 70 studies (n = 90,206) were included in qualitative synthesis. From these, 60 studies focused on MetS and LUTS: 44 reported positive correlations, 5 reported negative correlations, 11 reported no association, and 10 studies focused on MetS and total prostate volume (TPV). MetS positively correlated with moderate LUTS [odds ratio (OR) = 1.56, 95% confidence interval (CI) = 1.35-1.80], severe LUTS (OR = 2.35, 95% CI = 1.82-3.03), overactive bladder (OAB; OR = 3.2, 95% CI = 1.6-5.8), and nocturia severity (OR = 2.509, 95% CI = 1.571-4.007) at multivariate analysis. A total of 30 studies (n = 22,206) were included in meta-analysis; MetS was significantly associated with higher TPV (mean differences = 4.4450 ml, 95% CI = 2.0177-6.8723), but no significant predictive factors for effect sizes were discovered. CONCLUSION: Our meta-analysis demonstrates a significant association between the aggravating effects of MetS, which commonly coexists with obesity and benign prostate enlargement.

Vitamin D3 and its nuclear receptor increase the expression and activity of the human proton-coupled folate transporter.

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D(3) (vitamin D(3)) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D(3) ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-alpha (RXRalpha) in the presence of vitamin D(3). In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region -1694/-1680. DNA binding assays showed direct and specific binding of the VDR:RXRalpha heterodimer to the PCFT(-1694/-1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(-1694/-1680) motif mediates a transcriptional response to vitamin D(3). In functional support of this regulatory mechanism, treatment with vitamin D(3) significantly increased the uptake of [(3)H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D(3) and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D(3) may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

Non-linear dose-response of DNA-reactive genotoxins: recommendations for data analysis.

Until recently, there has only been a limited amount of data available on the kinetics of mutation induction in the low dose region of exposure. In our publication Doak et al. [S.H. Doak, G.J. Jenkins, G.E. Johnson, E. Quick, E.M. Parry, J.M. Parry, Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens, Cancer Res. 67 (2007) 3904-3911] we showed that the two alkylating agents methyl-methanesulfonate (MMS) and ethyl-methanesulfonate (EMS) possess non-linear dose-response curves with no observed effect levels (NOEL) for mutation or chromosomal damage in vitro. These experiments were carried out in the AHH-1 human lymphoblastoid cell line, using the hypoxanthine phosphoribosyl transferase (HPRT) assay and the cytokinesis-block micronucleus (CBMN) assay, respectively. We have now carried out more advanced statistical analyses to define threshold values, which is critical as it has a dramatic impact on hazard and risk assessment. To do this, we re-analysed the data to see if the linear model or a more complex model (hockey stick or quadratic) gave a significant better fit of the data. For both EMS and MMS cytokinesis-block micronucleus data sets, the hockey stick model gave the most significant fit. The same was true for EMS, MMS and surprisingly ethylnitrosourea (ENU) in the HPRT assay in human AHH-1 cells. However, methylnitrosourea (MNU) was linear in both assays. These further analyses have shown that EMS and MMS have clear thresholds for both gene mutation and chromosome damage, as does ENU for gene mutation in AHH-1 cells. MNU was linear for gene and chromosome mutation and so was ENU for chromosome mutations at the concentrations tested. These findings correlate closely with those in vivo findings of Gocke et al. [E. Gocke, L. Müller, In vivo studies in the mouse to define a threshold for the genotoxicity of EMS and ENU, Mutat. Res. (this issue)] and together these data show a true threshold for EMS both in vitro and in vivo. In this report, we will discuss the approaches that were taken to investigate potential threshold dose-response curves for DNA-reactive genotoxic compounds, with recommendations for further studies.

Influx transporter variants as predictors of cancer chemotherapy-induced toxicity: systematic review and meta-analysis.

AIM: Chemotherapeutic agents have been shown to increase lung patient survival, however their use may be limited by their serious adverse effects. We aimed to assess int impact of pharmacogenetic variation of influx transporters on inter-individual patient variation in adverse drug reactions. PATIENTS & METHODS: We conducted a meta-analysis and systemic review and identified 16 publications, totaling 1510 patients, to be eligible for review. RESULTS: Meta-analysis showed east-Asian patients expressing SLCO1B1 521T>C or 1118G>A to have a two- to fourfold increased risk of irinotecan-induced neutropenia but not diarrhea. American patients, expressing SLC19A1 IVS2(4935) G>A, were further associated with pemetrexed/gemcitabine-induced grade 3+ leukopenia. CONCLUSION: Future studies should look to robust validation of SLCO1B1 and SLC19A1 as prognostic markers in the management of lung cancer patients.

Efflux transporter variants as predictors of drug toxicity in lung cancer patients: systematic review and meta-analysis.

UNLABELLED: Chemotherapeutic drugs are underutilized in lung cancer management due in part to serious adverse drug reactions (ADRs). AIM: With studies revealing an association between interindividual patient ADR variation and efflux transporter variants, we carried out a meta-analysis and systemic review, in order to highlight current knowledge regarding the strength of association between efflux transporter SNPs variants and chemotherapeutic-drug induced ADRs. MATERIALS & METHODS: Papers were sourced from MEDLINE, Cochrane Library, CINHL, EMBASE, Web of Knowledge, Scopus. The Cochrane Collaboration Risk of Bias Tool v13 was used to evaluate six types of bias domains for each of the publications reviewed. RESULTS: Twenty-five publications comprising three randomised control trials, two retrospective case-controls and 20 clinical observation studies, totalling 3578 patients, were deemed eligible for review. Of the known efflux drug transporters, we report findings on the ABC members ABCB1, ABCC1, ABCC2, ABCG2, ABCA1, ABCC4 and ABCC5. Meta-analysis showed an decreased risk of irinotecan-induced neutropenia in patients expressing ABCB1 2677G>T/G (odds ratio [OR]: 0.24; 95% CI: 0.1-0.59; p = 0.002) but increased risk for ABCC2 3972T>T (OR: 1.67; 95% CI: 1.01-2.74; p = 0.04). ABCG2 34G>A was associated with a threefold increased risk of irinotecan-induced diarrhea (95% CI: 1.00-6.24; p = 0.05). CONCLUSION: The majority of studies have identified a role for variants in effluxdrug transporters in contributing to lung cancer treatment-associated ADRs. However, for implementation of use of these transporter genetic variants as prognostic markers for ADR risk, future studies must incorporate larger patient numbers.

Clinical Perspectives on Targeting Therapies for Personalized Medicine.

Expected benefits from new technology include more efficient patient selection for clinical trials, more cost-effective treatment pathways for patients and health services and a more profitable accelerated approach for drug developers. Regulatory authorities expect the pharmaceutical and biotechnology industries to accelerate their development of companion diagnostics and companion therapeutics toward the goal of safer and more effective personalized medicine, and expect health services to fund and prescribers to adopt these new therapeutic technologies. This review discusses the importance of a range of new approaches to developing new and reprofiled medicines to treat common and serious diseases, and rare diseases: new network pharmacology approaches, adaptive trial designs with enriched populations more likely to respond safely to treatment, as assessed by companion diagnostics for response and toxicity risk and use of "real world" data. Case studies are described of single and multiple protein drug targets in several important therapeutic areas. These case studies also illustrate the value and complexity of use of selective biomarkers of clinical response and risk of adverse drug effects, either singly or in combination.

Real-time reverse-transcription polymerase chain reaction: technical considerations for gene expression analysis.

The reverse transcription - polymerase chain reaction (RT-PCR) is a sensitive technique for the quantification of steady-state mRNA levels, particularly in samples with limited quantities of extracted RNA, or for analysis of low level transcripts. The procedure amplifies defined mRNA transcripts by taking advantage of retroviral enzymes with reverse transcriptase (RT) activity, coupled to PCR. The resultant PCR product concentration is directly proportional to the initial starting quantity of mRNA, therefore allowing quantification of gene expression by incorporation of a fluorescence detector for the appropriate amplicons. In this chapter, we describe a number of the most popular techniques for performing RT-PCR and detail the subsequent analysis methodologies required to interpret the resultant data in either a relative manner or through absolute quantification of gene expression levels.

N-methylpurine DNA glycosylase plays a pivotal role in the threshold response of ethyl methanesulfonate-induced chromosome damage.

Genotoxic tolerance to low-level exposure of monofunctional alkylating agents is compound specific, with the mechanism pertaining to alkyl-induced genotoxic threshold response as yet unknown. N-methylpurine DNA glycosylase (MPG), an initiator glycosylase of the base excision repair (BER) pathway, typically repairs alkyl-induced DNA adducts, many of which are associated with genomic instability and tumorigenic risk. Here we demonstrate the involvement of MPG in modulating the genotoxic threshold response induced by the Sn2 alkylating agent ethyl methanesulfonate (EMS) and not the Sn1 alkylating agent N-ethyl-N-nitrosourea (ENU) in human lymphoblastoid cells and suggest the lack of N7-ethylguanine adduct repair as a key factor attributable to an observed increase in EMS-induced chromosome damage. Moreover, an increase in MPG messenger RNA expression levels in response to EMS and not ENU doses administered below the low-observed effect level substantiates the proposed specific involvement of MPG in relation to EMS-induced genotoxicity. We further report an unexpected dose-dependent decrease in the mutation frequency of the MPG-deficient cell line M09B when challenged with ENU, a response deemed consequential to a pronounced dose-dependent increase in the number of apoptotic cells relative to wild type. Collectively, these findings implicate the differential involvement of MPG-directed BER as a primary mechanism of action for the chromosome damage threshold response and cytotoxicity induced by alkane sulfonates and N-nitrosourea compounds, respectively.

Pharmacogenetics of OATP (SLC21/SLCO), OAT and OCT (SLC22) and PEPT (SLC15) transporters in the intestine, liver and kidney.

The role of carrier-mediated transport in determining the pharmacokinetics of drugs has become increasingly evident with the discovery of genetic variants that affect expression and/or function of a given drug transporter. Drug transporters are expressed at numerous epithelial barriers, such as intestinal epithelial cells, hepatocytes, renal tubular cells and at the blood-brain barrier. Several recent studies have associated alterations in substrate uptake with the presence of SNPs. Here, we summarize the current knowledge on the functional and phenotypic consequences of genetic variation in intestinally, hepatically and renally expressed members of the organic anion-transporting polypeptide family (OATPs; SLC21/SLCO family), the organic anion and organic cation transporters (OATs/OCTs; SLC22 family) and the peptide transporter-1 (PEPT1; SLC15 family).