Prevalence of possible sarcopenia in community-dwelling older Chinese adults: a cross-sectional study.

OBJECTIVES: To determine the prevalence of possible sarcopenia and its association with other conditions in older adults in Bengbu, China. DESIGN, SETTING AND PARTICIPANTS: A cross-sectional study of 1082 community-dwelling Chinese people aged at least 60 years from March to June 2022. METHODS: Handgrip strength and information regarding associated conditions were collected. Possible sarcopenia was estimated based on handgrip strength with cut-off values (<28 kg in men; <18 kg in women) recommended by the Asia Working Group for Sarcopenia in 2019. Mann-Whitney U tests, χ2 tests and binary logistic regression analyses were used to explore relationships between possible sarcopenia and associated conditions. RESULTS: Possible sarcopenia was more prevalent in men (52.79%, n=246, age 79.43±7.33 years among men with possible sarcopenia) than in women (44.48%, n=274, age 78.90±7.71 years among women with possible sarcopenia). In men, possible sarcopenia positively correlated with high age (OR 2.658, 95% CI 1.758 to 4.019), physical inactivity (OR 2.779, 95% CI 1.646 to 4.691) and diabetes (OR 4.269, 95% CI 2.397 to 7.602), and negatively with hypertension (OR 0.586, 95% CI 0.384 to 0.893). The risk of possible sarcopenia in men decreased by 12.6% for every 1 kg/m2 increase of body mass index (OR 0.874, 95% CI 0.817 to 0.935). In women, possible sarcopenia positively correlated with high age (OR 3.821, 95% CI 2.677 to 5.455), physical inactivity (OR 2.185, 95% CI 1.488 to 3.210) and arthritis (OR 2.076, 95% CI 1.411 to 3.056). CONCLUSION: Possible sarcopenia is prevalent in older adults and the factors affecting possible sarcopenia are different in men and women. Health education about these target factors can be considered as a potential measure to prevent possible sarcopenia.

Green tea extract-induced lethal toxicity in fasted but not in nonfasted dogs.

Recent chronic toxicity studies performed on green tea extracts in fasted dogs have revealed some unique dose-limiting lethal liver, gastrointestinal, and renal toxicities. Key findings included necrosis of hepatic cells, gastrointestinal epithelia and renal tubules, atrophy of reproductive organs, atrophy and necrosis of hematopoietic tissues, and associated hematological changes. The polyphenol cachetins (a mixture of primarily epigallocatechin gallate [≥55%]; plus up to 10% each of epigallocatechin, epicatechin, and epigallocatechin gallate) appeared to be the causative agents for the observed toxicities because they are the active ingredients of green tea extract studied. Conduct of the study in nonfasted dogs under the same testing conditions and dose levels showed unremarkable results. Assuming both studies were valid, at the identified no observed adverse effect levels (NOAEL) of each study, systemic exposures (based on area under the curve [AUC]) were actually lower in fasted than nonfasted dogs, suggesting that fasting may have rendered the target organ systems potentially more vulnerable to the effects of green tea extract. The toxicity mechanisms that produced lethality are not known, but the results are scientifically intriguing. Because tea drinking has become more popular in the United States and abroad, the mode of action and site of action of green tea extract-induced lethal toxicities during fasting and the role of other phytochemical components of Folia Camellia sinensis (including nonpolyphenol fractions, which are often consumed when whole-leaf products are presented) warrant further investigation.

Altered apoptotic response in MCF 10A cells treated with the equine estrogen metabolite, 4-hydroxyequilenin.

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues including the breast. 4-Hydroxyequilenin (4-OHEN), a major catechol metabolite of equine estrogens present in Premarin, an estrogen replacement formulation, has been shown to induce apoptosis and DNA damage in human breast cancer cells. It also has the potential to be a tumor initiator or promoter and complete carcinogen. To further understand the effects and mechanisms of equine catechol estrogen metabolite 4-OHEN action in vitro, human non-tumorigenic mammary epithelial MCF 10A cell line was used to study the toxic effects of 4-OHEN. In this study, we observed that 4-OHEN caused dose-dependent increases in apoptosis and DNA damage as measured by the DAPI nuclear screening assay and the Comet assay, respectively. Interestingly, cells treated with 100 nM 4-OHEN biweekly for 4 weeks became resistant to cisplatin-induced apoptosis. The resistance to apoptosis of the 100 nM 4-OHEN-treated cells was through multiple regulatory mechanisms. Compared to the DMSO-treated cells, the 100 nM 4-OHEN-treated cells had higher GSH levels and total SOD activity, and a stronger GSH response after cisplatin treatment. Expression levels of several genes involved in cell growth, DNA repair, and apoptosis were either up- or down-regulated. These data indicate that long-term low-level equine estrogen metabolite exposure could induce DNA damage and initiate cells to become resistant to apoptosis.

Equine estrogen metabolite 4-hydroxyequilenin induces anchorage-independent growth of human mammary epithelial MCF-10A cells: differential gene expression.

Long-term exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. One potential mechanism of estrogen carcinogenesis involves catechol formation and these catechols are further oxidized to electrophilic/redox active o-quinones, which have the potential to both initiate and promote the carcinogenic process. Previously we showed that 4-hydroxyequilenin (4-OHEN) autoxidized to an o-quinone and caused a variety of damage to DNA. Since these deleterious effects could contribute to gene mutations, we investigated the Chinese hamster V79 cells to ascertain the relative ability of estradiol, 4-hydroxyestradiol, 17beta-hydroxyequilenin, 4,17beta-hydroxyequilenin, estrone, 4-hydroxyestrone, equilenin, and 4-hydroxyequilenin to induce the mutation of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene. All the 4-hydroxylated catechols induced significantly more colony formations in V79 cells as compared to the parent phenols at 100nM, suggesting that the catechol estrogen metabolites are more mutagenic towards the hprt gene than estrogens. Since 4-OHEN induced the highest mutation frequency, we examined a biomarker for transformation potential of this compound in MCF-10A cells using an anchorage-independent growth assay. Although 4-OHEN induced anchorage-independent growth of these cells, the isolated clones were not able to grow as tumors in vivo when injected into nude mice. These cells were assayed for genetic changes using cDNA microarrays. Real time RT-PCR confirmation of some of the differentially expressed genes showed down-regulation of metallothionein 2A, p53, BRCA1, and c-myc. Moreover, we showed the involvement of other genes important in cell transformation and oxidative stress, strengthening the hypothesis that this mechanism plays a considerable role in 4-OHEN-induced anchorage-independent growth.

Equine catechol estrogen 4-hydroxyequilenin is a more potent inhibitor of the variant form of catechol-O-methyltransferase.

Catechol-O-methyltransferase (COMT) plays an important role in the inactivation of biologically active and toxic catechols. It has been shown that COMT is genetically polymorphic with a wild-type and variant form where a valine has been substituted with a methionine. Several, but not all, epidemiological studies have shown that women, homozygous with the variant form, have an increased risk of developing breast cancer. Previously, we showed that 4-hydroxyequilenin (4-OHEN), a cytotoxic/genotoxic equine catechol estrogen metabolite, is both a substrate of COMT and an irreversible inhibitor of the methylation activity of COMT in vitro. To further understand the mechanism(s) of the association between the breast cancer risk and the COMT polymorphism, it was of interest to study the effect of the Val/Met polymorphism on COMT-catalyzed catechol estrogen methylation and 4-OHEN-mediated inhibition. In the present study, Michaelis-Menten analysis showed no difference between the relative ability of each form to methylate 4-OHEN. However, we found that the COMT variant form was more susceptible to 4-OHEN-mediated irreversible inactivation. Electrospray ionization mass spectrometry and SDS-gel analysis of COMT modified by 4-OHEN revealed that inhibition mechanisms include alkylation and/or oxidation of certain amino acids. In addition, site-directed mutagenesis experiments showed that Cys33 played a more important role in the variant form of COMT demonstrated by the fact that the C33A mutant of the variant form of COMT decreased its catalytic capability more dramatically as compared with that of wild type. Furthermore, thermotropic studies indicated that the variant form was more thermolabile, which suggested that the valine to methionine substitution may have changed the secondary/tertiary structure of the variant form of COMT, making it more susceptible to 4-OHEN and heat inactivation. These data suggest that 4-OHEN-mediated inhibition of the variant form of COMT in vivo might affect the detoxification efficiency of endogenous and/or exogenous catechol estrogens and play a role in the association between breast cancer risk and COMT polymorphism.

Antiestrogenic and DNA damaging effects induced by tamoxifen and toremifene metabolites.

The antiestrogen, tamoxifen, has been extensively used in the treatment and prevention of breast cancer. Although tamoxifen showed benefits in the chemotherapy and chemoprevention of breast cancer, epidemiological studies in both tamoxifen-treated breast cancer patients and healthy women indicated that treatment caused an increased risk of developing endometrial cancer. These troubling side effects lead to concerns over long-term safety of the drug. Therefore, it is important to fully understand the relationship between the antiestrogenic and the genotoxic mechanisms of tamoxifen, other antiestrogens, and their metabolites. Previously, we have shown that o-quinone formation from tamoxifen and its analogues, droloxifene and 4-hydroxytoremifene, may not contribute to the cytotoxic effects of these antiestrogens; however, these o-quinones can form adducts with deoxynucleosides and this implies that the o-quinone pathway could contribute to the genotoxicity of the antiestrogens in vivo. To further investigate this potential genotoxic pathway, we were interested in the role of estrogen receptor (ER)(1) alpha and beta since work with catechol estrogens has shown that ERs seem to enhance DNA damage in breast cancer cell lines. As a result, we investigated the binding affinities of 4-hydroxy and 3,4-dihydroxy derivatives of tamoxifen and toremifene to ER alpha and beta. The antiestrogenic activities of the metabolites using the Ishikawa cells were also investigated as well as their activity in ERalpha and ERbeta breast cancer cells using the transient transfection reporter, estrogen response element-dependent luciferase assay. The data showed that the antiestrogenic activities of these compounds in the biological assays mimicked their activities in the ER binding assay. To determine if the compounds were toxic and if ERs played a role in this process, the cytotoxicity of these compounds in ERbeta41(2) (ERbeta), S30 (ERalpha), and MDA-MB-231 (ER(-)) cell lines was compared. The results showed that the cytotoxicity differences between the metabolites were modest. In addition, all of the metabolites showed similar toxicity patterns in both ER positive and negative cell lines, which means that the ER may not contribute to the cytotoxicity pathway. Finally, we compared the amount of DNA damage induced by these metabolites in these cell lines using the comet assay. The catechols 3,4-dihydroxytoremifene and 3,4-dihydroxytamoxifen induced a greater amount of cellular single strand DNA cleavage as compared with the phenols in all cell lines. The different amounts of DNA damage in ER positive and negative cell lines suggested that the ERs might play a role in this process. These data suggest that the formation of catechols represents a minor role in cytotoxic and antiestrogenic effects in cells as compared with their phenol analogues. However, catechols induced more DNA damage at nontoxic doses in breast cancer cells, which implies that o-quinones formed from catechols could contribute to genotoxicity in vivo, which is ER-dependent.

Effect of halogenated substituents on the metabolism and estrogenic effects of the equine estrogen, equilenin.

Estrogen replacement therapy has been correlated with an increased risk for developing breast and endometrial cancers. One potential mechanism of estrogen carcinogenesis involves metabolism of estrogens to 2- and 4-hydroxylated catechols, which are further oxidized to electrophilic/redox active o-quinones that have the potential to both initiate and promote the carcinogenic process. Previously, we showed that the equine estrogens, equilin and equilenin, which are major components of the estrogen replacement formulation Premarin (Wyeth-Ayerst), are primarily metabolized to the catechol, 4-hydroxyequilenin. This catechol was found to autoxidize to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. To block catechol formation from equilenin, 4-halogenated equilenin derivatives were synthesized. These derivatives were tested for their ability to bind to the estrogen receptor, induce estrogen sensitive genes, and their potential to form catechol metabolites. We found that the 4-fluoro derivatives were more estrogenic than the 4-chloro and 4-bromo derivatives as demonstrated by a higher binding affinity for estrogen receptors alpha and beta, an enhanced induction of alkaline phosphatase activity in Ishikawa cells, pS2 expression in S30 cells, and PR expression in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than the parent compounds, equilenin and 17beta-hydroxyequilenin. In addition, these halogenated compounds showed less cytotoxicity in the presence of tyrosinase than the parent compounds in S30 cells. Also, as stated above, the 4-fluoro derivatives showed similar estrogenic effects as compared with parent compounds; however, they were less toxic in S30 cells as compared to equilenin and 17beta-equilenin. Because 17beta-hydroxy-4-halogenated equilenin derivatives showed higher estrogenic effects than the halogenated equilenin derivatives in vitro, we studied the relative ability of the 17beta-hydroxy-4-halogenated equilenin derivatives to induce estrogenic effects in the ovariectomized rat model. The 4-fluoro derivative showed higher activity than 4-chloro and 4-bromo derivatives as demonstrated by inducing higher vaginal cellular differentiation, uterine growth, and mammary gland branching. However, 17beta-hydroxy-4-fluoroequilenin showed a lower estrogenic activity than 17beta-hydroxyequilenin and estradiol, which could be due to alternative pharmacokinetic properties for these compounds. These data suggest that the 4-fluoroequilenin derivatives have promise as alternatives to traditional estrogen replacement therapy due to their similar estrogenic properties with less overall toxicity.

Catechol estrogen 4-hydroxyequilenin is a substrate and an inhibitor of catechol-O-methyltransferase.

Redox and/or electrophilic metabolites formed during estrogen metabolism may play a role in estrogen carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is the major phase I catechol metabolite of the equine estrogens equilenin and equilin, which are components of the most widely prescribed estrogen replacement formulation, Premarin. Previously, we have found that 4-OHEN rapidly autoxidized to an o-quinone in vitro and caused toxic effects such as the inactivation of human detoxification enzymes. 4-OHEN has also been shown to be a substrate for catechol-O-methyltransferase (COMT) in human breast cancer cells. In the present study, we demonstrated that 4-OHEN was not only a substrate of recombinant human soluble COMT in vitro with a K(m) of 2.4 microM and k(cat) of 6.0 min(-)(1) but it also inhibited its own methylation by COMT at higher concentrations in the presence of the reducing agent dithiothreitol. In addition, 4-OHEN was found to be an irreversible inhibitor of COMT-catalyzed methylation of the endogenous catechol estrogen 4-hydroxyestradiol with a K(i) of 26.0 microM and a k(2) of 1.62 x 10(-)(2) s(-)(1). 4-OHEN in vitro not only caused the formation of intermolecular disulfide bonds as demonstrated by gel electrophoresis, but electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry also showed that 4-OHEN alkylated multiple residues of COMT. Peptide mapping experiments further indicated that Cys33 in recombinant human soluble COMT was the residue most likely modified by 4-OHEN in vitro. These data suggest that inhibition of COMT methylation by 4-OHEN might reduce endogenous catechol estrogen clearance in vivo and further enhance toxicity.

RPA phosphorylation in mitosis alters DNA binding and protein-protein interactions.

The heterotrimeric DNA-binding protein, replication protein A (RPA), consists of 70-, 34-, and 14-kDa subunits and is involved in maintaining genomic stability by playing key roles in DNA replication, repair, and recombination. RPA participates in these processes through its interaction with other proteins and its strong affinity for single-stranded DNA (ssDNA). RPA-p34 is phosphorylated in a cell-cycle-dependent fashion primarily at Ser-29 and Ser-23, which are consensus sites for Cdc2 cyclin-dependent kinase. By systematically examining RPA-p34 phosphorylation throughout the cell cycle, we have found there are distinct phosphorylated forms of RPA-p34 in different cell-cycle stages. We have isolated and purified a unique phosphorylated form of RPA that is specifically associated with the mitotic phase of the cell cycle. The mitotic form of RPA (m-hRPA) shows no difference in ssDNA binding activity as compared with recombinant RPA (r-hRPA), yet binds less efficiently to double-stranded DNA (dsDNA). These data suggest that mitotic phosphorylation of RPA-p34 inhibits the destabilization of dsDNA by RPA complex, thereby decreasing the binding affinity for dsDNA. The m-hRPA also exhibits altered interactions with certain DNA replication and repair proteins. Using highly purified proteins, m-hRPA exhibited decreased binding to ATM, DNA pol alpha, and DNA-PK as compared to unphosphorylated recombinant RPA (r-hRPA). Dephosphorylation of m-hRPA was able to restore the interaction with each of these proteins. Interestingly, the interaction of RPA with XPA was not altered by RPA phosphorylation. These data suggest that phosphorylation of RPA-p34 plays an important role in regulating RPA functions in DNA metabolism by altering specific protein-protein interactions.

Inhibition of cellular enzymes by equine catechol estrogens in human breast cancer cells: specificity for glutathione S-transferase P1-1.

Glutathione S-transferases (GSTs) are a family of detoxification isozymes that protect cells by conjugating GSH to a variety of toxic compounds, and they may also play a role in the regulation of both cellular proliferation and apoptosis. We have previously shown that human GST P1-1, which is the most widely distributed extrahepatic isozyme, could be inactivated by the catechol estrogen metabolite 4-hydroxyequilenin (4-OHEN) in vitro [Chang, M., Shin, Y. G., van Breemen, R. B., Blond, S. Y., and Bolton, J. L. (2001) Biochemistry 40, 4811-4820]. In the present study, we found that 4-OHEN and another catechol estrogen, 4,17beta-hydroxyequilenin (4,17beta-OHEN), significantly decreased GSH levels and the activity of GST within minutes in both estrogen receptor (ER) negative (MDA-MB-231) and ER positive (S30) human breast cancer cells. In addition, 4-OHEN caused significant decreases in GST activity in nontransformed human breast epithelial cells (MCF-10A) but not in the human hepatoma HepG2 cells, which lack GST P1-1. We also showed that GSH partially protected the inactivation of GST P1-1 by 4-OHEN in vitro, and depletion of cellular GSH enhanced the 4-OHEN-induced inhibition of GST activity. In addition, 4-OHEN GSH conjugates contributed about 27% of the inactivation of GST P1-1 by 4-OEHN in vitro. Our in vitro kinetic inhibition experiments with 4-OHEN showed that GST P1-1 had a lower K(i) value (20.8 microM) compared to glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 52.4 microM), P450 reductase (PR, 77.4 microM), pyruvate kinase (PK, 159 microM), glutathione reductase (GR, 230 microM), superoxide dismutase (SOD, 448 microM), catalase (562 microM), GST M1-1 (620 microM), thioredoxin reductase (TR, 694 microM), and glutathione peroxidase (GPX, 1410 microM). In contrast to the significant inhibition of total GST activity in these human breast cancer cells, 4-OHEN only slightly inhibited the cellular GAPDH activity, and other cellular enzymes including PR, PK, GR, SOD, catalase, TR, and GPX were resistant to 4-OHEN-induced inhibition. These data suggest that GST P1-1 may be a preferred protein target for equine catechol estrogens in vivo.

Oxidative DNA damage induced by equine estrogen metabolites: role of estrogen receptor alpha.

Excessive exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. 4-Hydroxyequilenin (4-OHEN), a major metabolite of equine estrogens present in estrogen replacement formulations, has been shown to induce cytotoxic/carcinogenic effects. In the present study, we have found that 4-OHEN caused DNA damage in breast cancer cells, and cells that contain estrogen receptor alpha (S30) are more sensitive to 4-OHEN-mediated DNA damage as compared to estrogen receptor negative cells (MDA-MB-231). For example, concentration-dependent increases in 8-oxo-deoxyguanosine (8-oxo-dG), as measured by LC-MS-MS or by the Fpg comet assay, were only detected in the S30 cells, and the amount of this lesion could be enhanced by agents, which catalyze redox cycling (NADH) or deplete GSH (diethyl maleate). The role of the estrogen receptor in modulating DNA damage was further established in incubations with the ER antagonist tamoxifen, where decreases in 8-oxo-deoxyguanosine were observed. Another equine estrogen metabolite, 4,17 beta-hydroxyequilenin (4,17 beta-OHEN), was found to have the same cytotoxicity and a similar ability to induce reactive oxygen species (ROS), and caused the same oxidative DNA damage in S30 cells as compared to 4-OHEN. However, 4,17 beta-OHEN induced twice as much single strand DNA breaks in S30 cells compared to 4-OHEN. Also 4,17 beta-OHEN was more estrogenic than 4-OHEN as demonstrated by a higher binding affinity for ER alpha and an enhanced induction in activity of estrogen-dependent alkaline phosphatase in Ishikawa cells. These data suggest that the mechanism of DNA damage induced by equine estrogen metabolites could involve oxidative stress and that the estrogen receptor may play a role in this process.