Clastogenic and mutagenic effects of bisphenol A: an endocrine disruptor

Bisphenol A (BPA) is a well-known endocrine disruptor (ED) which represents a major toxicological and public health concern due to its widespread exposure to humans. BPA has been reported to induce DNA adduct and aneuploidy in rodents. Recent studies in humans depicted its association with recurrent miscarriages and male infertility due to sperm DNA damage indicating that BPA might have genotoxic activity. Hence, the present study was designed to determine genotoxic and mutagenic effects of BPA using in-vivo and in-vitro assays. The adult male and female rats were orally administered with various doses of BPA (2.4 μg, 10 μg, 5mg and 50mg/kgbw) once a day for six consecutive days. Animals were sacrificed, bone marrow and blood samples were collected and subjected to series of genotoxicity assay such as micronucleus, chromosome aberration and single cell gel electrophoresis (SCGE) assay respectively. Mutagenicity was determined using tester strains of Salmonella typhimurium (TA 98, TA 100 and TA 102) in the presence and absence of metabolically active microsomal fractions (S9). Further, we estimated the levels of 8-hydroxydeoxyguanosine, lipid per-oxidation and glutathione activity to decipher the potential genotoxic mechanism of BPA. We observed that BPA exposure caused a significant increase in the frequency of micronucleus (MN) in polychromatic erythrocytes (PCEs), structural chromosome aberrations in bone marrow cells and DNA damage in blood lymphocytes. These effects were observed at various doses tested except 2.4 μg compared to vehicle control. We did not observe the mutagenic response in any of the tester strains tested at different concentrations of BPA. We found an increase in the level of 8-hydroxydeoxyguanosine in the plasma and increase in lipid per-oxidation and decrease in glutathione activity in liver of rats respectively which were exposed to BPA. In conclusion, the data obtained clearly documents that BPA is not mutagenic but exhibit genotoxic activity and oxidative stress could be one of the mechanisms leading to genetic toxicity.     

Aged‐senescent cells contribute to impaired heart regeneration

Aging leads to increased cellular senescence and is associated with decreased potency of tissue‐specific stem/progenitor cells. Here, we have done an extensive analysis of cardiac progenitor cells (CPCs) isolated from human subjects with cardiovascular disease, aged 32–86 years. In aged subjects (>70 years old), over half of CPCs are senescent (p16^INK4A, SA‐β‐gal, DNA damage γH2AX, telomere length, senescence‐associated secretory phenotype [SASP]), unable to replicate, differentiate, regenerate or restore cardiac function following transplantation into the infarcted heart. SASP factors secreted by senescent CPCs renders otherwise healthy CPCs to senescence. Elimination of senescent CPCs using senolytics abrogates the SASP and its debilitative effect in vitro. Global elimination of senescent cells in aged mice (INK‐ATTAC or wild‐type mice treated with D + Q senolytics) in vivo activates resident CPCs and increased the number of small Ki67‐, EdU‐positive cardiomyocytes. Therapeutic approaches that eliminate senescent cells may alleviate cardiac deterioration with aging and restore the regenerative capacity of the heart.     

Secretory function of autophagy in innate immune cells

Eukaryotic cells utilize two main secretory pathways to transport proteins to the extracellular space. Proteins with a leader signal sequence often undergo co‐translational transport into the endoplasmic reticulum (ER), and then to the Golgi apparatus before they reach their destination. This pathway is called the conventional secretory pathway. Proteins without signal peptides can bypass this ER‐Golgi system and are secreted by a variety of mechanisms collectively called the unconventional secretory pathway. The molecular mechanisms of unconventional secretion are emerging. Autophagy is a conserved bulk degradation mechanism that regulates many intracellular functions. Recent evidence implicates autophagy in the secretory pathway. This review focuses on potential secretory roles of autophagy and how they could modulate the functions of innate immune cells that secrete a wide range of mediators in response to environmental and biological stimuli. We provide a brief overview of the secretory pathways, enumerate the potential mechanistic themes by which autophagy interacts with these pathways and describe their relevance in the context of innate immune cell function.     

Alterations in collagen metabolism and increased fibroproliferation in the heart in cerium-treated rats implications for the pathogenesis of endomyocardial fibrosis

Cerium (Ce), a rare earth element, has been postulated to play a role in the pathogenesis of tropical endomyocardial fibrosis (EMF). Investigations carried out recently in pursuance of the postulation furnished histological evidence of EMF and increased cardiac collagen content in rats on prolonged administration of Ce. The present study was undertaken to understand the molecular basis of myocardial injury and fibrosis produced by the element. This article presents evidence of increased lipid peroxidation and elevated rates of fibroblast proliferation and collagen deposition in the heart in Ce-treated rats. It is suggested that the element may trigger a wound-healing response in the cardiac tissue leading to cardiac fibrosis.     

Natural Indoles,Indole-3-Carbinol (I3C) and 3,3’-Diindolylmethane (DIM), Attenuate Staphylococcal Enterotoxin B-Mediated Liver Injury by Downregulating miR-31 Expression and Promoting Caspase-2-Mediated Apoptosis

Staphylococcal enterotoxin B (SEB) is a potent superantigen capable of inducing inflammation characterized by robust immune cell activation and proinflammatory cytokine release. Exposure to SEB can result in food poisoning as well as fatal conditions such as toxic shock syndrome. In the current study, we investigated the effect of natural indoles including indole-3-carbinol (I3C) and 3,3’-diindolylmethane (DIM) on SEB-mediated liver injury. Injection of SEB into D-galactosamine-sensitized female C57BL/6 mice resulted in liver injury as indicated by an increase in enzyme aspartate transaminase (AST) levels, induction of inflammatory cytokines, and massive infiltration of immune cells into the liver. Administration of I3C and DIM (40mg/kg), by intraperitonal injection, attenuated SEB-induced acute liver injury, as evidenced by decrease in AST levels, inflammatory cytokines and cellular infiltration in the liver. I3C and DIM triggered apoptosis in SEB-activated T cells primarily through activation of the intrinsic mitochondrial pathway. In addition, inhibitor studies involving caspases revealed that I3C and DIM-mediated apoptosis in these activated cells was dependent on caspase-2 but independent of caspase-8, 9 and 3. In addition, I3C and DIM caused a decrease in Bcl-2 expression. Both compounds also down-regulated miR-31, which directly targets caspase-2 and influences apoptosis in SEB-activated cells. Our data demonstrate for the first time that indoles can effectively suppress acute hepatic inflammation caused by SEB and that this may be mediated by decreased expression of miR-31 and consequent caspase-2-dependent apoptosis in T cells.     

Influence of gestation on jugular plasma thyroxine levels in Murrah buffalo and Karan Swiss cows

Plasma samples from 106 pregnant Karan Swiss (Brown Swiss x Sahiwal) cows and 108 Murrah buffalo were tested for thyroxine (T(4)) levels to determine the relationship between the hormonal changes and advancing pregnancy in the two species. All samples were collected within 2 months (January and February) to avoid seasonal interference on T(4) levels. In pregnant cows, the concentration of T(4) increased sharply during the first trimester, reaching a peak at the third month of gestation followed by a gradual decline until the last month of pregnancy. In pregnant buffalo, peripheral plasma T(4) levels fluctuated slightly throughout pregnancy without exhibiting a specific trend. Statistical analysis revealed that the magnitude of T(4) levels was significantly lower in buffalo (P < 0.01) than in the cows throughout pregnancy and that the hormonal patterns of the two species were significantly different (P < 0.05) during gestation. It was hypothesized from this study that T(4) requirements for the fetal buffalo calf may be lower than that for the fetal cattle calf since the buffalo gestation period is a month longer and the metabolic rate lower vis-a-vis the cow.     

Structure determination and biochemical studies on Bacillus stearothermophilus E53Q serine hydroxymethyltransferase and its complexes provide insights on function and enzyme memory

Serine hydroxymethyltransferase (SHMT) belongs to the alpha-family of pyridoxal 5'-phosphate-dependent enzymes and catalyzes the reversible conversion of L-Ser and tetrahydrofolate to Gly and 5,10-methylene tetrahydrofolate. 5,10-Methylene tetrahydrofolate serves as a source of one-carbon fragment in many biological processes. SHMT also catalyzes the tetrahydrofolate-independent conversion of L-allo-Thr to Gly and acetaldehyde. The crystal structure of Bacillus stearothermophilus SHMT (bsSHMT) suggested that E53 interacts with the substrate, L-Ser and tetrahydrofolate. To elucidate the role of E53, it was mutated to Q and structural and biochemical studies were carried out with the mutant enzyme. The internal aldimine structure of E53QbsSHMT was similar to that of the wild-type enzyme, except for significant changes at Q53, Y60 and Y61. The carboxyl of Gly and side chain of L-Ser were in two conformations in the respective external aldimine structures. The mutant enzyme was completely inactive for tetrahydrofolate-dependent cleavage of L-Ser, whereas there was a 1.5-fold increase in the rate of tetrahydrofolate-independent reaction with L-allo-Thr. The results obtained from these studies suggest that E53 plays an essential role in tetrahydrofolate/5-formyl tetrahydrofolate binding and in the proper positioning of Cbeta of L-Ser for direct attack by N5 of tetrahydrofolate. Most interestingly, the structure of the complex obtained by cocrystallization of E53QbsSHMT with Gly and 5-formyl tetrahydrofolate revealed the gem-diamine form of pyridoxal 5'-phosphate bound to Gly and active site Lys. However, density for 5-formyl tetrahydrofolate was not observed. Gly carboxylate was in a single conformation, whereas pyridoxal 5'-phosphate had two distinct conformations. The differences between the structures of this complex and Gly external aldimine suggest that the changes induced by initial binding of 5-formyl tetrahydrofolate are retained even though 5-formyl tetrahydrofolate is absent in the final structure. Spectral studies carried out with this mutant enzyme also suggest that 5-formyl tetrahydrofolate binds to the E53QbsSHMT-Gly complex forming a quinonoid intermediate and falls off within 4 h of dialysis, leaving behind the mutant enzyme in the gem-diamine form. This is the first report to provide direct evidence for enzyme memory based on the crystal structure of enzyme complexes.     

Identification and characterization of a hitherto unknown nucleotide-binding domain and an intricate interdomain regulation in HflX-a ribosome binding GTPase

A role for HflX in 50S-biogenesis was suggested based on its similarity to other GTPases involved in this process. It possesses a G-domain, flanked by uncharacterized N- and C-terminal domains. Intriguingly, Escherichia coli HflX was shown to hydrolyze both GTP and adenosine triphosphate (ATP), and it was unclear whether G-domain alone would explain ATP hydrolysis too. Here, based on structural bioinformatics analysis, we suspected the possible existence of an additional nucleotide-binding domain (ND1) at the N-terminus. Biochemical studies affirm that this domain is capable of hydrolyzing ATP and GTP. Surprisingly, not only ND1 but also the G-domain (ND2) can hydrolyze GTP and ATP too. Further; we recognize that ND1 and ND2 influence each other’s hydrolysis activities via two salt bridges, i.e. E29-R257 and Q28-N207. It appears that the salt bridges are important in clamping the two NTPase domains together; disrupting these unfastens ND1 and ND2 and invokes domain movements. Kinetic studies suggest an important but complex regulation of the hydrolysis activities of ND1 and ND2. Overall, we identify, two separate nucleotide-binding domains possessing both ATP and GTP hydrolysis activities, coupled with an intricate inter-domain regulation for Escherichia coli HflX.