Can sustained exposure to PFAS trigger a genotoxic response? A comprehensive genotoxicity assessment in mice after subacute oral administration of PFOA and PFBA.

PFAS (perfluoroalkyl substances) are considered non-genotoxic. However, PFAS exposure has been associated with the induction of oxidative stress in vitro and in vivo, and the possible induction of indirect genotoxic effects under sustained PFAS exposure has not been investigated. In order to shed light on this aspect, in this study a comprehensive assessment of genotoxicity was carried out in mice administered with perfluorooctanoic acid (PFOA, 0.1, 1 and 5 mg/kg body weight) and its C4 analogue perfluorobutyric acid (PFBA, 5 mg/kg body weight) for five weeks through drinking water. Markers of cell toxicity, oxidative stress and DNA strand breaks were measured in liver, the main target of toxicity of PFOA in rodents; systemic genotoxicity was also assessed by the analysis of micronuclei in reticulocytes and spleen lymphocytes, and germ cell effects by the Comet assay on testis cells. PFOA administration at the highest dose (5 mg/kg body weight) induced marked liver hypertrophy with signs of cell injury (elevated ALT and AST), with no concurrent evidence of lipid peroxidation and oxidative stress (decreased antioxidant capacity). Only mild liver hypertrophy, with no other signs of toxicity, was determined by PFBA administration. No evidence of treatment related genotoxicity was observed in any experimental group. Overall, data indicate that under the experimental conditions of this study, severe liver toxicity induced by PFOA administration is not associated with oxidative stress. Accordingly, no genotoxic effect is observed in liver and in the other tissues examined. Milder evidence of liver toxicity, with no genotoxicity, and a lower tendency to bioaccumulation were observed in PFBA treated mice.

DNA damage response in monozygotic twins discordant for smoking habits.

Previous studies in twins indicate that non-shared environment, beyond genetic factors, contributes substantially to individual variation in mutagen sensitivity; however, the role of specific causative factors (e.g. tobacco smoke, diet) was not elucidated. In this investigation, a population of 22 couples of monozygotic twins with discordant smoking habits was selected with the aim of evaluating the influence of tobacco smoke on individual response to DNA damage. The study design virtually eliminated the contribution of genetic heterogeneity to the intra-pair variation in DNA damage response, and thus any difference in the end-points investigated could directly be attributed to the non-shared environment experienced by co-twins, which included as main factor cigarette smoke exposure. Peripheral lymphocytes of study subjects were challenged ex vivo with γ-rays, and the induction, processing, fixation of DNA damage evaluated through multiple approaches. Folate status of study subjects was considered significant covariate since it is affected by smoking habits and can influence radiosensitivity. Similar responses were elicited by γ-rays in co-twins for all the end-points analysed, despite their discordant smoking habits. Folate status did not modify DNA damage response, even though a combined effect of smoking habits, low-plasma folic acid level, and ionising radiation was observed on apoptosis. A possible modulation of DNA damage response by duration and intensity of tobacco smoke exposure was suggested by Comet assay and micronucleus data, but the effect was quantitatively limited. Overall, the results obtained indicate that differences in smoking habits do not contribute to a large extent to inter-individual variability in the response to radiation-induced DNA damage observed in healthy human populations.

Toxic and genotoxic effects of oral administration of furan in mouse liver.

In this study, the effects induced in mouse liver by repeated oral exposure to furan were investigated. To this aim, the compound was given for 28 days by daily gavage to male B6C3F1 mice at 2, 4, 8 and 15 mg/kg body weight (b.w.)/day. Twenty-four hours after last administration, animals were sacrificed, liver was excised and the following parameters were evaluated: histological alterations, apoptosis, cell proliferation, polyploidy, overall DNA methylation, gene expression and DNA damage by the immunofluorescence detection of foci of phosphorylated histone H2AX (gamma-H2AX) and by alkaline comet assays, using both standard and modified protocols for the detection of DNA cross links. Liver DNA damage by comet assays was also evaluated in mice receiving furan as a single acute oral dose (15, 100 or 250 mg/kg b.w.). Microscopic analysis of liver sections indicated that repeated oral administration of furan was moderately toxic, producing mild histological alterations with necrotic figures, apoptosis and limited regenerative cell proliferation. The flow cytometric analysis of DNA content in single-cell suspensions of liver cells showed a statistically significant increase in polyploid (8N) cells at the highest dose. No treatment-related changes in overall DNA methylation, gamma-H2AX foci, DNA strand breaks and cross links were observed at the end of the 4-week exposure period. However, several genes involved in DNA damage response, beyond stress and liver toxicity, were over-expressed in mice treated with the highest furan dose (15 mg/kg b.w./day). Acute administration of furan induced evident liver toxicity at the highest dose (250 mg/kg b.w.), which was associated with a significant increase of DNA damage in the alkaline comet assay and with a distinct decrease in gamma-ray-induced DNA migration. Overall, the results obtained suggest that the contribution of genotoxicity to the mechanism of furan carcinogenicity in mouse liver should not be dismissed.

Influence of family history of type 2 diabetes on leptin concentration in cord blood of male offspring with high birth weight.

To verify whether a diabetes family history might be a risk factor for the development, in adult age, of metabolic disorders, leptin, anthropometric and endocrine parameters were analysed in 95 babies with grandparents affected by type 2 diabetes (DF) and in 95 matched babies without diabetes family history (NDF). A sexual dimorphism for leptin was present in the NDF group (males: 6.7+/-4.1 ng/ml; females: 12.3+/-6.5; p < 0.0001) but not in the DF group (males: 9.0+/-5.5; females: 10.8+/-6.4), due to the significant increase in DF male leptin level, compared to that of NDF males (p < 0.05). In DF males only, leptin was positively correlated with body length, PI, C-peptide, IGF-1 and IGF1BP3. These results suggest that the increase in DF male leptin could be a compensatory mechanism for reduced insulin sensitivity in a pre-clinical alteration of glucose metabolism.

Effects of folic acid deficiency and MTHFR C677T polymorphism on spontaneous and radiation-induced micronuclei in human lymphocytes.

Folic acid plays a key role in the maintenance of genomic stability, providing methyl groups for the conversion of uracil to thymine and for DNA methylation. Besides dietary habits, folic acid metabolism is influenced by genetic polymorphism. The C677T polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene is associated with a reduction of catalytic activity and is suggested to modify cancer risk differently depending on folate status. In this work the effect of folic acid deficiency on genome stability and radiosensitivity has been investigated in cultured lymphocytes of 12 subjects with different MTHFR genotype (four for each genotype). Cells were grown for 9 days with 12, 24 and 120 nM folic acid and analyzed in a comprehensive micronucleus test coupled with centromere characterization by CREST immunostaining. In other experiments, cells were grown with various folic acid concentrations, irradiated with 0.5 Gy of gamma rays and analyzed in the micronucleus test. The results obtained indicate that folic acid deficiency induces to a comparable extent chromosome loss and breakage, irrespective of the MTHFR genotype. The effect of folic acid was highly significant (P < 0.001) and explained >50% of variance of both types of micronuclei. Also nucleoplasmic bridges and buds were significantly increased under low folate supply; the increase in bridges was mainly observed in TT cells, highlighting a significant effect of the MTHFR genotype (P = 0.006) on this biomarker. Folic acid concentration significantly affected radiation-induced micronuclei (P < 0.001): the increased incidence of radiation-induced micronuclei with low folic acid was mainly accounted for by carriers of the variant MTHFR allele (both homozygotes and heterozygotes), but the overall effect of genotype did not attain statistical significance. Treatment with ionizing radiations also increased the frequency of nucleoplasmic bridges. The effect of folic acid level on this end-point was modulated by the MTHFR genotype (P for interaction = 0.02), with TT cells grown at low folic acid concentration apparently resistant to the induction of radiation-induced bridges. Finally, the effect of in vitro folate deprivation on global DNA methylation was evaluated in lymphocytes of six homozygous subjects (three CC and three TT). The results obtained suggest that, under the conditions of this work, folic acid deprivation is associated with global DNA hypermethylation.

Overweight and metabolic and hormonal parameter disruption are induced in adult male mice by manipulations during lactation period.

Neonatal manipulations (10 min of maternal separation plus s.c. sham injection, daily for the first 21 d of life) determine overweight in male adult mice. In this work, we investigated the mechanisms underlying mild obesity and the alteration of caloric balance. Neonatally manipulated mice become overweight after onset of maturity, showing increased fat tissue and hypertrophic epididymal adipocytes. Increase in body weight occurs in the presence of a small increase in daily food intake (significant only in the adult period) and the absence of a decrease in spontaneous locomotor activity, while the calculated caloric efficiency is higher in manipulated mice, especially in adulthood. Fasting adult animals show hyperglycemia, hyperinsulinemia, hypertriglyceridemia, hypercholesterolemia, and hyperleptinemia. Soon after weaning and in the adulthood, plasma corticosterone and adrenocorticotropin (ACTH) are also significantly increased. Thus, neonatal manipulations in nongenetically susceptible male mice program mild obesity, with metabolic and hormonal alterations that are similar to those found in experimental models of diabetes mellitus, suggesting that this metabolic derangement may have at least part of its roots early on in life and, more interestingly, that psychological and nociceptive stimuli induce these features.

Vasoactive intestinal peptide (VIP): a new neuroendocrine marker of clinical progression in chronic heart failure?

OBJECTIVE: Vasoactive intestinal peptide (VIP) is a powerful vasodilatory neuropeptide with positive inotropic and chronotropic properties. The aim of the study was to investigate the pathophysiological role of VIP in heart failure. DESIGN AND RESULTS: VIP was assayed in plasma within the first in-hospital day in 52 patients with heart failure due to dilated cardiomyopathy. The concentration of VIP was: (i) higher in patients than in healthy subjects; (ii) higher in elderly but not in younger patients compared with healthy controls; (iii) inversely related to NYHA class: higher in NYHA 2 than in NYHA > 2 patients and in normal subjects, in both young and elderly groups; (iv) not correlated with echocardiographic parameters and (v) not influenced by the aetiology of dilated cardiomyopathy. CONCLUSIONS: The physiological properties of VIP suggest that the increased plasma concentrations in patients with heart failure contribute to restore the compromised haemodynamic balance either by improving myocardial performance or by counteracting the harmful effects related to simultaneous activation of other neuroendocrine systems, i.e. the sympathetic and renin-angiotensin systems. Decreased VIP concentrations are related to progressive worsening of heart failure. The higher VIP concentrations in elderly patients compared with healthy controls suggest that the capacity to increase VIP production is preserved in older people.

Mutational analysis of StAR gene in adrenal tumors.

Adrenal adenomas and carcinomas are mostly monoclonal, suggesting that a genetic alteration in a progenitor cell may contribute to their development. However, the molecular pathogenesis of these tumors still remains unclear. It has been already excluded that activating mutations of the ACTH receptor or of G protein stimulator alpha sub-units, affecting cAMP pathway, is involved in the tumorigenesis. Therefore, this work has been focused on post-transductional (ACTH) signal alterations and in particular on the mutational analysis of the Steroid Acute Regulatory protein (StAR) gene to verify whether somatic mutations or genomic polymorphisms of this gene may be correlated with adrenal tumorigenesis. Tissue DNA was extracted from 40 functional and non-functional adrenocortical tumors that were removed from patients aged between 17 and 72 years (mean 43 +/- 4). Blood DNA was obtained from 24 patients (aged between 26 and 70 years) affected by adrenal tumors and from 100 healthy subjects without radiological and clinical evidence of adrenal masses, aged between 25-35 years (90 Caucasians and 10 Africans). The DNA was used as the template for the amplification of the StAR gene using the polymerase chain reaction. The amplified DNA of each exon of the StAR gene was purified and sequenced in automatic sequenciator. With the exception of exon 5 showing in codon 203 an homozygous missense mutation, the sequence of the other exons of the StAR gene resulted normal in all tumors studied. The same homozygous mutation (Asp203Ala) was observed in the sequence of exon 5 performed on genomic DNA of the 24 affected patients and in the control subjects. The homozygosity of the mutation observed in all patients (either in tissue or blood samples) and in control subjects, independently of their ethnic origin, led us to suggest that the Asp203Ala cannot be considered as mutation or as polymorphism, but that it must be considered as a mistake in the sequence entered in the Genbank, which needs to be modified accordingly. These data, and those up to now reported in the literature, allow us to suggest that mutations of the gene coding for the protein involved in the initial step of the steroidogenesis could not be considered as a possible cause for the development of adrenal tumors.