Impact of Alcohol on Bone Health in People Living With HIV: Integrating Clinical Data From Serum Bone Markers With Morphometric Analysis in a Non-Human Primate Model.

People living with HIV (PLWH) represent a vulnerable population to adverse musculoskeletal outcomes due to HIV infection, antiretroviral therapy (ART), and at-risk alcohol use. Developing measures to prevent skeletal degeneration in this group requires a grasp of the relationship between alcohol use and low bone mass in both the PLWH population and its constituents as defined by sex, age, and race. We examined the association of alcohol use with serum biochemical markers of bone health in a diverse cohort of PLWH enrolled in the New Orleans Alcohol Use in HIV (NOAH) study. To explore the effects of alcohol on bone in the context of HIV and ART and the role of estrogen, we conducted a parallel, translational study using simian immunodeficiency virus (SIV)+/ART+ female rhesus macaques divided into four groups: vehicle (Veh)/Sham; chronic binge alcohol (CBA)/Sham; Veh/ovariectomy (OVX); and CBA/OVX. Clinical data showed that both osteocalcin (Ocn) and procollagen type I N-propeptide (PINP) levels were inversely associated with multiple measures of alcohol consumption. Age (>50 years) significantly increased susceptibility to alcohol-associated suppression of bone formation in both female and male PLWH, with postmenopausal status appearing as an additional risk factor in females. Serum sclerostin (Scl) levels correlated positively with measures of alcohol use and negatively with Ocn. Micro-CT analysis of the macaque tibias revealed that although both CBA and OVX independently decreased trabecular number and bone mineral density, only OVX decreased trabecular bone volume fraction and impacted cortical geometry. The clinical data implicate circulating Scl in the pathogenesis of alcohol-induced osteopenia and suggest that bone morphology can be significantly altered in the absence of net change in osteoblast function as measured by serum markers. Inclusion of sophisticated tools to evaluate skeletal strength in clinical populations will be essential to understand the impact of alcohol-induced changes in bone microarchitecture. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

EB 2017 Article: Soy protein isolate feeding does not result in reproductive toxicity in the pre-pubertal rat testis.

The isoflavone phytoestrogens found in the soy protein isolate used in soy infant formulas have been shown to have estrogenic actions in the developing male reproductive tract resulting in reproductive toxicity. However, few studies have examined potential estrogenicity of soy protein isolate as opposed to that of pure isoflavones. In this study, we fed weanling male Sprague-Dawley rats a semi-purified diet with casein or soy protein isolate as the sole protein source from postnatal day 21 to 33. Additional groups were fed casein or soy protein isolate and treated s.c. with 10 µg/kg/d estradiol via osmotic minipump. Estradiol treatment reduced testis, prostate weights, and serum androgen concentrations ( P < 0.05). Soy protein isolate had no effect. Estradiol up-regulated 489 and down-regulated 1237 testicular genes >1.5-fold ( P < 0.05). In contrast, soy protein isolate only significantly up-regulated expression of 162 genes and down-regulated 16 genes. The top 30 soy protein isolate-up-regulated genes shared 93% concordance with estradiol up-regulated genes. There was little overlap between soy protein isolate down-regulated genes and those down-regulated by estradiol treatment. Functional annotation analysis revealed significant differences in testicular biological processes affected by estradiol or soy protein isolate. Estradiol had major actions on genes involved in reproductive processes including down-regulation of testicular steroid synthesis and expression of steroid receptor activated receptor (Star) and cytochrome P450 17α-hydroxylase/(Cyp17a1). In contrast, soy protein isolate primarily affected pathways associated with macromolecule modifications including ubiquitination and histone methylation. Our results indicate that rather than acting as a weak estrogen in the developing testis, soy protein isolate appears to act as a selective estrogen receptor modulator with little effect on reproductive processes. Impact statement Soy protein isolate (SPI) is the sole protein used to make soy-based infant formulas. SPI contains phytoestrogens, which are structurally similar to estradiol. These phytoestrogens, daidzein, genistein, and equol, fit the definition of endocrine-disrupting compounds, and at high concentrations, have estrogenic actions resulting in reproductive toxicity in the developing male, when provided as isolated chemicals. However, few animal studies have examined the potential estrogenicity of SPI as opposed to pure isoflavones. In this study, SPI feeding did not elicit an estrogenic response in the testis nor any adverse outcomes including reduced testicular growth, or androgen production during early development in rats when compared to those receiving estradiol. These findings are consistent with emerging data showing no differences in reproductive development in males and female children that received breast milk, cow's milk formula, or soy infant formula during the postnatal feeding period.

Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet.

Alcoholic and nonalcoholic fatty liver diseases are risk factors for development of hepatocellular carcinoma, but the underlying mechanisms are poorly understood. On the other hand, ingestion of soy-containing diets may oppose the development of certain cancers. We previously reported that replacing casein with a soy protein isolate reduced tumor promotion in the livers of mice with alcoholic liver disease after feeding a high fat ethanol liquid diet following initiation with diethylnitrosamine. Feeding soy protein isolate inhibited processes that may contribute to tumor promotion including inflammation, sphingolipid signaling, and Wnt/ß-catenin signaling. We have extended these studies to characterize liver tumor promotion in a model of nonalcoholic fatty liver disease produced by chronic feeding of high-fat liquid diets in the absence of ethanol. Mice treated with diethylnitrosamine on postnatal day 14 were fed a high-fat liquid diet made with casein or SPI as the sole protein source for 16 weeks in adulthood. Relative to mice fed normal chow, a high fat/casein diet led to increased tumor promotion, hepatocyte proliferation, steatosis, and inflammation. Replacing casein with soy protein isolate counteracted these effects. The high fat diets also resulted in a general increase in transcripts for Wnt/ß-catenin pathway components, which may be an important mechanism, whereby hepatic tumorigenesis is promoted. However, soy protein isolate did not block Wnt signaling in this nonalcoholic fatty liver disease model. We conclude that replacing casein with soy protein isolate blocks development of steatosis, inflammation, and tumor promotion in diethylnitrosamine-treated mice fed high fat diets. Impact statement The impact of dietary components on cancer is a topic of great interest for both the general public and the scientific community. Liver cancer is currently the second leading form of cancer deaths worldwide. Our study has addressed the effect of the protein source on hepatic tumor promotion in a mouse model reflecting aspects of non-alcoholic fatty liver disease (NAFLD). A high-fat liquid diet with casein as the protein source promotes hepatic injury and tumor promotion in diethylnitrosamine-treated mice. Replacing casein with a soy protein isolate led to a pronounced diminishment of tumor promotion and associated hepatic injury and inflammation. The study thus demonstrates that a dietary protein source can have beneficial, preventative effects on hepatic tumor promotion.

Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples.

High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt.

Mammary gland morphology and gene expression signature of weanling male and female rats following exposure to exogenous estradiol.

In order to characterize the actions of xenoestrogens, it is essential to possess a solid portrait of the physiological effects of exogenous estradiol. We assessed effects of three doses of exogenous estradiol (E2) (0.1, 1.0 and 10 µg/kg/day) given between postnatal days 21 and 33 on the mammary gland morphology and gene expression profiles of male and female rats compared to vehicle-treated controls. The male mammary gland was more responsive to E2 treatment than in females, with 509 genes regulated >2-fold in a dose-dependent manner in males and only 174 in females. In males, E2 treatment significantly (P < 0.01) increased the number of terminal end buds (TEBs) and the expression of proliferating cell nuclear antigen (PCNA) protein (P < 0.05), both of which are indicators of proliferation. This change was linked to a significant increase (P < 0.05) in the expression of the gene encoding amphiregulin, which is known to induce TEB formation. There was also a dose-dependent increase (P < 0.001) in the estrogen-regulated gene encoding the progesterone receptor. In intact females, despite lack of changes in mammary morphology, we observed a dose-dependent increase (P < 0.05) in the expression of genes encoding three milk proteins: whey acidic protein, casein beta and casein kappa. There was a significant (P < 0.05) downregulation of both estrogen receptors in response to E2 treatment. These results suggest that mammary glands of male rats are very sensitive to exogenous E2 during development post-weaning. The dose-dependent increase observed in amphiregulin and progesterone receptor gene expression was linked to morphological changes and represents a reliable and sensitive tool to evaluate estrogenicity. In contrast, intact weanling female rats were less responsive.