High-content imaging analyses of the effects of bisphenols and organophosphate esters on TM4 mouse Sertoli cells†.

The endocrine disruptive effects of bisphenol A (BPA) and brominated flame retardants (BDE-47) have led to restrictions on their use and increased the pressure to identify safe replacements for these chemicals. Although there is evidence that some of these alternatives may be toxic to spermatogonial and Leydig cells, little is known about the toxicity of emerging replacements on Sertoli cells. We used high-content imaging to compare the effects of legacy chemicals, BPA and BDE-47, to their corresponding replacements. TM4 Sertoli cells were exposed for 48 h to each chemical (0.001-100 µM) followed by cytotoxicity and phenotypic endpoint assessment. The benchmark concentration potency ranking for bisphenols based on cytotoxicity was BPTMC > bisphenol M > BPAF>BPF > BPS > BPA. Human administered equivalent dose (AED) determination ranked BPS as the most potent alternative replacement. The benchmark concentration potency ranking of BDE-47 and organophosphate esters based on cytotoxicity was TDtBPP>BDMPP>TBOEP>TDCPP>TMPP>TPHP>BDE47>IPPP=BPDP=TCPP. Additionally, TM4 cell exposure to BDE-47 increased Calcein intensity (57.9 µM) and affected lysosomes (21.6 µM), while exposure to TPHP and TMPP resulted in cellular oxidative stress changes at benchmark concentration values as low as 0.01 and 0.4 µM, respectively. Overall bioactivity considerations of the chemicals on TM4 via ToxPi analyses and AED modeling further validated emerging replacements as highly potent chemicals in comparison to BPA and BDE-47. These findings demonstrate that many bisphenol and flame retardant replacements are more potent in Sertoli cells than the legacy chemical they are replacing and that phenotypic parameter assessment is an effective tool in chemical toxicity assessment.

Phthalates and alternative plasticizers differentially affect phenotypic parameters in gonadal somatic and germ cell lines†.

The developmental and reproductive toxicity associated with exposure to phthalates has motivated a search for alternatives. However, there is limited knowledge regarding the adverse effects of some of these chemicals. We used high-content imaging to compare the effects of mono (2-ethylhexyl) phthalate (MEHP) with six alternative plasticizers: di-2-ethylhexyl terephthalate (DEHTP); diisononyl-phthalate (DINP); di-isononylcyclohexane-1,2-dicarboxylate (DINCH); 2-ethylhexyl adipate (DEHA); 2,2,4-trimethyl 1,3-pentanediol diisobutyrate (TXIB) and di-iso-decyl-adipate (DIDA). A male germ spermatogonial cell line (C18-4), a Sertoli cell line (TM4) and two steroidogenic cell lines (MA-10 Leydig and KGN granulosa) were exposed for 48 h to each chemical (0.001-100 µM). Cell images were analyzed to assess cytotoxicity and effects on phenotypic endpoints. Only MEHP (100 µM) was cytotoxic and only in C18-4 cells. However, several plasticizers had distinct phenotypic effects in all four cell lines. DINP increased Calcein intensity in C18-4 cells, whereas DIDA induced oxidative stress. In TM4 cells, MEHP, and DINCH affected lipid droplet numbers, while DEHTP and DINCH increased oxidative stress. In MA-10 cells, MEHP increased lipid droplet areas and oxidative stress; DINP decreased the number of lysosomes, while DINP, DEHA, and DIDA altered mitochondrial activity. In KGN cells, MEHP, DINP and DINCH increased the number of lipid droplets, whereas DINP decreased the number of lysosomes, increased oxidative stress and affected mitochondria. The Toxicological Priority Index (ToxPi) provided a visual illustration of the cell line specificity of the effects on phenotypic parameters. The lowest administered equivalent doses were observed for MEHP. We propose that this approach may assist in screening alternative plasticizers.

Acyl-CoA synthetase long-chain 5 genotype is associated with body composition changes in response to lifestyle interventions in postmenopausal women with overweight and obesity: a genetic association study on cohorts Montréal-Ottawa New Emerging Team, and Complications Associated with Obesity.

BACKGROUND: Genetic studies on Acyl-CoA Synthetase Long-Chain 5 (ACSL5) demonstrate an association between rs2419621 genotype and rate of weight loss in women with obesity in response to caloric restriction. Our objectives were to (1) confirm results in two different populations of women with overweight and obesity (2) study rs2419621's influence on body composition parameters of women with overweight and obesity following lifestyle interventions. METHODS: rs2419621 genotype was determined in women with overweight and obesity who participated in the Montréal-Ottawa New Emerging Team (MONET n = 137) and Complications Associated with Obesity (CAO n = 37) studies. Genotyping was done using TaqMan MGB probe-based assay. Multiple linear regression analyses were used to test for associations. RESULTS: When studying women with overweight and obesity, rs2419621 [T] allele carriers had a significantly greater decrease in visceral fat, absolute and percent fat mass and a greater increase in percent lean mass in response to lifestyle intervention in comparison to non-carriers. Studying only individuals with obesity showed similar results with rs2419621 [T] allele carriers also displaying a significantly greater decrease in body mass index following the lifestyle intervention in comparison to non-carriers. CONCLUSION: Women with overweight and obesity carrying the ACSL5 rs2419621 [T] allele are more responsive to lifestyle interventions in comparison to non-carriers. Conducting such genetic association studies can aid in individualized treatments/interventions catered towards an individual's genotype.

Enediynyl peptides and iso-coumarinyl methyl sulfones as inhibitors of proprotein convertases PCSK8/SKI-1/S1P and PCSK4/PC4: Design, synthesis and biological evaluations.

The proprotein convertases PCSK8 and PCSK4 are, respectively, the 8th and 4th members of Ca(+2)-dependent serine endoprotease of Proprotein Convertase Subtilisin Kexin (PCSK) super family structurally related to the bacterial subtilisin and yeast kexin. The membrane bound PCSK8 (also called SKI-1 or S1P) is implicated in sterol regulation and lipid synthesis via its role in the maturation of human (h) SREBP-2. It also plays role in cartilage formation, bone mineralization, as well as viral pathogenesis. On the other hand, PCSK4 has been linked to mammalian fertilization and placenta growth. Owing to these findings, interest has grown to develop specific inhibitors against these enzymes for potential biochemical and therapeutic applications. In this study we developed two types of small molecule inhibitors of PCSK8 and PCSK4 and demonstrated their anti-proteolytic activities in vitro cell-free and in vitro cell culture systems. These are isocoumarinyl methyl sulfone derivatives and enediyne amino acid containing peptides. Our in vitro data suggested that one of the 7 sulfone derivatives (methyl phenyl sulfone) inhibited PCSK8 with inhibition constant Ki ∼255µM. It also blocked PCSK8-mediated processing of hSREBP-2 in HepG2 cell in a concentration-dependent manner. However all 7 iso-coumarinyl methyl sulfones inhibited htrypsin with IC50 ranging from 2 to 165µM. In contrast, all our designed enediynyl peptides inhibited PCSK8 and PCSK4 activity with Ki and IC50 in low µM or high nM ranges. All compounds exhibited competitive inhibition as indicated by their enzyme kinetic plots and observed dependence of IC50 value on substrate concentration. Our study confirmed that incorporation at the substrate cleavage site of 'Enediyne amino acid' generates potent inhibitors of PCSK8 and PCSK4. This represents a novel approach for future development of inhibitors of PCSK or other enzymes.

Elucidation of the Effects of Bisphenol A and Structural Analogs on Germ and Steroidogenic Cells Using Single Cell High-Content Imaging.

Concerns about the potential adverse effects of bisphenol A (BPA) have led to an increase in the use of replacements, yet the toxicity data for several of these chemicals are limited. Using high-content imaging, we compared the effects of BPA, BPAF, BPF, BPS, BPM, and BPTMC in germ (C18-4 spermatogonial) and steroidogenic (MA-10 Leydig and KGN granulosa) cell lines. Effects on cell viability and phenotypic markers were analyzed to determine benchmark concentrations (BMCs) and estimate administered equivalent doses (AEDs). In all 3 cell lines, BPA was one of the least cytotoxic bisphenol compounds tested, whereas BPM and BPTMC were the most cytotoxic. Interestingly, BPF and BPS were cytotoxic only in MA-10 cells. Effects on phenotypic parameters, including mitochondria, lysosomes, lipid droplets, and oxidative stress, were both bisphenol- and cell-line specific. BPA exposure affected mitochondria (BMC: 1.2 µM; AED: 0.09 mg/kg/day) in C18-4 cells. Lysosome numbers were increased in MA-10 cells exposed to BPA or BPAF but decreased in KGN cells exposed to BPAF or BPM. Lipid droplets were decreased in C18-4 cells exposed to BPF and in MA-10 cells exposed to BPTMC but increased in BPF, BPM, and BPTMC-exposed KGN cells. BPA and BPM exposure induced oxidative stress in MA-10 and KGN cells, respectively. In summary, structurally similar bisphenols displayed clear cell-line-specific differences in BMC and AED values for effects on cell viability and phenotypic endpoints. This approach, together with additional data on human exposure, may aid in the selection and prioritization of responsible replacements for BPA. .

ACSL5 genotype influence on fatty acid metabolism: a cellular, tissue, and whole-body study.

BACKGROUND: Acyl-CoA Synthetase Long Chain 5 (ACSL5) gene's rs2419621 T/C polymorphism was associated with ACSL5 mRNA expression and response to lifestyle interventions. However, the mechanistic understanding of the increased response in T allele carriers is lacking. Study objectives were to investigate the effect of rs2419621 genotype and ACSL5 human protein isoforms on fatty acid oxidation and respiration. METHODS: Human ACSL5 overexpression in C2C12 mouse myoblasts was conducted to measure 14C palmitic acid oxidation and protein isoform localization in vitro. 14C palmitic acid oxidation studies and Western blot analysis of ACSL5 proteins were carried out in rectus abdominis primary myotubes from 5 rs2419621 T allele carriers and 4 non-carriers. In addition, mitochondrial high-resolution respirometry was conducted on vastus lateralis muscle biopsies from 4 rs2419621 T allele carriers and 4 non-carriers. Multiple linear regression analysis was conducted to test the association between rs2419621 genotype and respiratory quotient related pre- and post-lifestyle intervention measurements in postmenopausal women with overweight or obesity. RESULTS: In comparison to rs2419621 non-carriers, T allele carriers displayed higher levels of i) 683aa ACSL5 isoform, localized mainly in the mitochondria, playing a greater role in fatty acid oxidation in comparison to the 739aa protein isoform ii) in vitro CO2 production in rectus abdominis primary myotubes iii) in vivo fatty acid oxidation and lower carbohydrate oxidation post-intervention iv) ex vivo complex I and II tissue respiration in vastus lateralis muscle. CONCLUSIONS: These results support the conclusion that rs2419621 T allele carriers, are more responsive to lifestyle interventions partly due to an increase in the short ACSL5 protein isoform, increasing cellular, tissue and whole-body fatty acid utilization. With the increasing effort to develop personalized medicine to combat obesity, our findings provide additional insight into genotypes that can significantly affect whole body metabolism and response to lifestyle interventions.