Preadipocyte stimulating factor in rat serum: evidence for a discrete 63 kDa protein that promotes cell differentiation of rat preadipocytes in primary cultures

In primary cultures of rat preadipocytes (PA) isolated from epididymal or perirenal depots, rat serum is more effective than other animal sera (fetal calf, newborn calf, human, horse, rabbit, cat, sheep, goat, dog, pig) in promoting adipogenic conversion, biochemical differentiation, and mitogenesis. Only mouse serum is comparable to rat serum. This activity is attributable to a specific growth factor (preadipocyte stimulating factor, PSF). An assay for PSF in rat serum was devised using PA from perirenal fat of 3-month-old Fischer 344 rats grown first to confluence in FCS for 8 days and then for the next 3 days in test serum, followed by measurement of triglyceride (TG) and glycerol-3-phosphate dehydrogenase (GPDH). Rat serum induces dose-dependent rapid cell division, which coincides with accumulation of TG and increase of GPDH; for routine quantitation, TG is assayed. The biochemical characteristics of PSF in serum are as follows: stable at 4 degrees C for up to 1 year; inactivated at 100 degrees C (80% loss, 30 min) but stable at 56 degrees C for 1 hr; stable at pH 2-12; non-dialyzable; completely resistant to pepsin, trypsin, and chymotrypsin but destroyed by pronase and subtilisn; stable to DTT and periodate; and m.w. between 68 kDa (Sephacryl-300) and 58 kDa (Sephacryl-300 in 5 M urea). PSF activity is greater in serum from Wistar than from Fischer 344 rats, while activity of serum from Zucker obese (fa/fa) rats is at least as great as that from Wistar rats and, like serum of rats made obese by feeding a high-fat, high-carbohydrate diet, is not suppressed. PSF activity is not due to insulin, insulin-like growth factor-1 (IGF-1), growth hormone, glucocorticoids, or combinations of these hormones. PSF activity was not seen with a number of growth factors including colony-stimulating factor (CSF-1), GM-CSF, interleukins 1, 2, and 3, neuroleukin, tumor necrosis factor, and others. PSF is distinct from the low molecular weight (4-8 kDa) differentiation factor present in rat serum, FCS, and human serum that promotes the adipogenic conversion and cellular differentiation of 3T3-L1, 3T3-F442A, and Ob17 cells. PSF appears to be a new differentiation factor for rat preadipocytes, has properties suggestive of a highly glycosylated protein, and may be highly species specific.     

Identification of lipopolysaccharide-binding proteins in 70Z/3 cells by photoaffinity cross-linking

A radioiodinated, photoactivatable derivative of Salmonella minnesota Re595 lipopolysaccharide (LPS) was used to label LPS-binding proteins in 70Z/3 cells. The labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by autoradiography. 125I-Labeled-2-(p-azidosalycylamido)1,3'-dithiopropionamide S. minnesota Re595 LPS (125I-ASD-Re595) labeled a limited number of proteins. The most prominent of these had a apparent molecular mass of 18 kDa. Less prominent labeling of 25- and 28-kDa proteins was also seen. Labeling was saturated by 5 micrograms/ml 125I-ASD-Re595 and was inhibited by a 10-100-fold excess of unlabeled LPS or lipid A. Labeling was maximal within 30 min at 37 degrees C; much less labeling occurred at lower temperatures. The proteins labeled with 125I-ASD-Re595 appear to be on the surface of the cell, since they can be digested by trypsin and were found in the membrane fraction of the cell but not in the cytosol. Studies with competitive inhibitors suggested that the proteins bind to the lipid A region of the LPS molecule. Biologically inactive lipid A analogs were poor inhibitors of labeling, suggesting that the LPS-binding proteins could discriminate between active lipid A and inactive analogs. These studies suggest that the 18- and 25-kDa proteins bind specifically to the lipid A region of the LPS molecule and should be considered as candidates for a functional LPS receptor.     

Changes measured in arterial blood following a single breath of cigarette smoke in rabbits.

This paper describes a method for monitoring short term changes in arterial blood in rabbits in response to a single breath of cigarette smoke. The method was developed to investigate the observation that neutrophil transit times through the lung are extended during acute exposures to cigarette smoke (1). In this model, we sought to monitor the time course of appearance of diffusible gas from smoke to the blood stream, the appearance of lipid peroxidation products and the activation of neutrophils. New Zealand white rabbits were anesthetized and fitted with a tracheostomy tube and an aortic catheter. Smoke was collected in a syringe from a non-filtered cigarette and injected immediately via the tracheostomy tube. Blood samples were collected at 1 second intervals. Carboxyhemoglobin levels increased 108% over pre-smoke levels, peaking at 5-7 seconds after the start of smoke exposure. Serum conjugated dienes, as measured by change in absorbance of lipid extracts at 234 nm, increased 40%, peaking at 10-11 seconds. Thiobarbituric acid (TBA) reactive material exhibited a variable response, with a statistically insignificant maximum at 12 seconds. Serum myeloperoxidase activity was not affected by smoke inhalation. This method provides a model for studying the acute effects of smoke inhalation and provides some evidence for oxidant stress following a single breath of cigarette smoke.     

An investigation of the photo-clastogenic potential of ultrafine titanium dioxide particles

Ultrafine titanium dioxide is widely used in a number of commercial products including sunscreens and cosmetics. There is extensive evidence on the safety of ultrafine titanium dioxide. However, there are some published studies indicating that some forms at least may be photogenotoxic, photocatalytic and/or carcinogenic. In order to clarify the conflicting opinions on the safety of ultrafine titanium dioxide particles, the current studies were performed to investigate the photo-clastogenic potential of eight different classes of ultrafine titanium dioxide particles. The photo-clastogenicity of titanium dioxide was measured in Chinese hamster ovary (CHO) cells in the absence and presence of UV light at a dose of 750 mJ/cm². The treatments were short (3 h) followed by a 17-h recovery and achieved concentrations that either induced approximately 50% cytotoxicity or reached 5000 μg/ml if non-cytotoxic. None of the titanium dioxide particles tested induced any increase in chromosomal aberration frequencies either in the absence or presence of UV. These studies show that ultrafine titanium dioxide particles do not exhibit photochemical genotoxicity in the model system used.     

The pathogen reduction treatment of platelets with S-59 HCl (Amotosalen) plus ultraviolet A light: Genotoxicity profile and hazard assessment

Despite restrictive donor criteria and screening procedures, infections resulting from the transfusion of bacterially contaminated platelet products continue to occur. Pathogen reduction technologies targeting nucleic acids have been developed. However, concerns about the safety of these procedures exist; the main concern being the possible mutagenic and carcinogenic effects of the pathogen-inactivated preparation in the recipient. This report reviews the genotoxicity profile of the S-59 (Amotosalen) plus long wavelength ultraviolet light (UVA) pathogen reduction technology, and assesses the mutagenic and carcinogenic hazards in recipients of treated platelets. S-59, a synthetic heterocyclic psoralen, non-covalently intercalates into the nucleic acids of pathogens and forms crosslinks when UVA photoactivated. Before clinical use, the levels of residual S-59 and free photoproducts are greatly reduced using a ‘compound adsorption device’ (CAD). In vitro, S-59 is mutagenic in Salmonella typhimurium and mouse lymphoma L5178Y TK^+/− cells, and is clastogenic in CHO cells. There is reduced activity (Salmonella, CHO cells) or no activity (mouse lymphoma cells) with metabolic activation (S9 mix). When tested up to toxic dose levels, S-59 was negative in the mouse bone marrow micronucleus assay and the rat hepatocyte unscheduled DNA synthesis (UDS) test. Based on comparative studies conducted with S-59 plus UVA-treated platelets (up to 25 times without CAD), any genotoxic effects can be attributed to residual S-59. Considering (1) the known genotoxic mechanism of action for S-59, (2) the negative in vivo studies for S-59 at multiples >40,000× over clinical peak plasma levels, and (3) the fact that the positive in vitro genotoxicity effects for the end product seem due to residual S-59, any mutagenic hazard to a recipient of S-59 plus UVA-treated platelets is negligible and there is no concern about a carcinogenic potential as a consequence of a mutagenic activity. This conclusion is supported by a negative p53^+/− mouse carcinogenicity study.     

Mendelian randomization supports causality between maternal hyperglycemia and epigenetic regulation of leptin gene in newborns

Leptin is an adipokine that acts in the central nervous system and regulates energy balance. Animal models and human observational studies have suggested that leptin surge in the perinatal period has a critical role in programming long-term risk of obesity. In utero exposure to maternal hyperglycemia has been associated with increased risk of obesity later in life. Epigenetic mechanisms are suspected to be involved in fetal programming of long term metabolic diseases. We investigated whether DNA methylation levels near LEP locus mediate the relation between maternal glycemia and neonatal leptin levels using the 2-step epigenetic Mendelian randomization approach. We used data and samples from up to 485 mother-child dyads from Gen3G, a large prospective population-based cohort. First, we built a genetic risk score to capture maternal glycemia based on 10 known glycemic genetic variants (GRS₁₀) and showed it was an adequate instrumental variable (β = 0.046 mmol/L of maternal fasting glucose per additional risk allele; SE = 0.007; P = 7.8 × 10⁻¹¹; N = 467). A higher GRS₁₀ was associated with lower methylation levels at cg12083122 located near LEP (β = −0.072 unit per additional risk allele; SE = 0.04; P = 0.05; N = 166). Direction and effect size of association between the instrumental variable GRS₁₀ and methylation at cg12083122 were consistent with the negative association we observed using measured maternal glycemia. Lower DNA methylation levels at cg12083122 were associated with higher cord blood leptin levels (β = −0.17 log of cord blood leptin per unit; SE = 0.07; P = 0.01; N = 170). Our study supports that maternal glycemia is part of causal pathways influencing offspring leptin epigenetic regulation.     

Niacin requirements for genomic stability

Through its involvement in over 400 NAD(P)-dependent reactions, niacin status has the potential to influence every area of metabolism. Niacin deficiency has been linked to genomic instability largely through impaired function of the poly ADP-ribose polymerase (PARP) family of enzymes. In various models, niacin deficiency has been found to cause impaired cell cycle arrest and apoptosis, delayed DNA excision repair, accumulation of single and double strand breaks, chromosomal breakage, telomere erosion and cancer development. Rat models suggest that most aspects of genomic instability are minimized by the recommended levels of niacin found in AIN-93 formulations; however, some beneficial responses do occur in the range from adequate up to pharmacological niacin intakes. Mouse models show a wide range of protection against UV-induced skin cancer well into pharmacological levels of niacin intake. It is currently a challenge to compare animal and human data to estimate the role of niacin status in the risk of genomic instability in human populations. It seems fairly certain that some portion of even affluent populations will benefit from niacin supplementation, and some subpopulations are likely well below an optimal intake of this vitamin. With exposure to stressors, like chemotherapy or excess sunlight, suraphysiological doses of niacin may be beneficial.