Rescue of Methionine Dependence by Cobalamin in a Human Colorectal Cancer Cell Line.

Methionine dependence is a characteristic of most cancer cells where they are unable to proliferate when the essential amino acid methionine is replaced with its precursor homocysteine in the growing media. Normal cells, on the other hand, thrive under these conditions and are referred to as methionine-independent. The reaction that adds a methyl group from 5-methyltetrahydrofolate to homocysteine to regenerate methionine is catalyzed by the enzyme methionine synthase with the cofactor cobalamin (vitamin B12). However, decades of research have shown that methionine dependence in cancer is not due to a defect in the activity of methionine synthase. Cobalamin metabolism has been tied to the dependent phenotype in rare cell lines. We have identified a human colorectal cancer cell line in which the cells regain the ability to proliferation in methionine-free, L-homocystine-supplemented media when cyanocobalamin is supplemented at a level of 1 µg/mL. In human SW48 cells, methionine replacement with L-homocystine does not induce any measurable increase in apoptosis or reactive oxygen species production in this cell line. Rather, proliferation is halted, then restored in the presence of cyanocobalamin. Our data show that supplementation with cyanocobalamin prevents the activation of the integrated stress response (ISR) in methionine-deprived media in this cell line. The ISR-associated cell cycle arrest, characteristic of methionine-dependence in cancer, is also prevented, leading to the continuation of proliferation in methionine-deprived SW48 cells with cobalamin. Our results highlight differences between cancer cell lines in the response to cobalamin supplementation in the context of methionine dependence.

Differences in cell death in methionine versus cysteine depletion.

Restriction of the sulfur amino acids methionine and cysteine has recently been proposed as potential adjuvant therapy in cancer. While cysteine depletion has been associated with ferroptotic cell death, methionine depletion has not. We hypothesized that comparing the response of melanoma cell lines to depletion of the amino acids methionine and cysteine would give us insight into the critical role in cancer of these two closely related amino acids. We analyzed the response to three conditions: methionine depletion, methionine replacement with homocysteine, and cysteine depletion. In cancer cells, the transcription factor ATF4 was induced by all three tested conditions. The replacement of methionine with homocysteine produced a strong ferroptotic gene signature. We also detected an activation of the NRF2 antioxidant pathway by both methionine and cysteine depletion. Total glutathione levels were decreased by 42% in melanoma cells grown without methionine, and by 95% in cells grown without cysteine. Lipid peroxidation was increased in cells grown without cysteine, but not in cells grown without methionine. Despite the large degree of overlap in gene expression between methionine and cysteine depletion, methionine depletion and replacement of methionine with homocysteine was associated with apoptosis while cysteine depletion was associated with ferroptosis. Glutamine depletion produced comparable gene expression patterns and was associated with a 28% decrease in glutathione. Apoptosis was detected in these cells. In this experiment, a strong ATF4-driven ferroptotic gene signature was insufficient to induce ferroptosis without a concomitant profound decrease in glutathione levels.

Methionine dietary supplementation potentiates ionizing radiation-induced gastrointestinal syndrome.

Methionine is an essential amino acid needed for a variety of processes in living organisms. Ionizing radiation depletes tissue methionine concentrations and leads to the loss of DNA methylation and decreased synthesis of glutathione. In this study, we aimed to investigate the effects of methionine dietary supplementation in CBA/CaJ mice after exposure to doses ranging from 3 to 8.5 Gy of 137Cs of total body irradiation. We report that mice fed a methionine-supplemented diet (MSD; 19.5 vs. 6.5 mg/kg in a methionine-adequate diet, MAD) developed acute radiation toxicity at doses as low as 3 Gy. Partial body irradiation performed with hindlimb shielding resulted in a 50% mortality rate in MSD-fed mice exposed to 8.5 Gy, suggesting prevalence of radiation-induced gastrointestinal syndrome in the development of acute radiation toxicity. Analysis of the intestinal microbiome demonstrated shifts in the gut ecology, observed along with the development of leaky gut syndrome and bacterial translocation into the liver. Normal gut physiology impairment was facilitated by alterations in the one-carbon metabolism pathway and was exhibited as decreases in circulating citrulline levels mirrored by decreased intestinal mucosal surface area and the number of surviving crypts. In conclusion, we demonstrate that a relevant excess of methionine dietary intake exacerbates the detrimental effects of exposure to ionizing radiation in the small intestine.NEW & NOTEWORTHY Methionine supplementation, instead of an anticipated health-promoting effect, sensitizes mice to gastrointestinal radiation syndrome. Mechanistically, excess of methionine negatively affects intestinal ecology, leading to a cascade of physiological, biochemical, and molecular alterations that impair normal gut response to a clinically relevant genotoxic stressor. These findings speak toward increasing the role of registered dietitians during cancer therapy and the necessity of a solid scientific background behind the sales of dietary supplements and claims regarding their benefits.

Decaffeinated Green Tea Extract Does Not Elicit Hepatotoxic Effects and Modulates the Gut Microbiome in Lean B6C3F1 Mice.

The main purpose of this study was to investigate the hepatotoxic potential and effects on the gut microbiome of decaffeinated green tea extract (dGTE) in lean B6C3F1 mice. Gavaging dGTE over a range of 1X-10X mouse equivalent doses (MED) for up to two weeks did not elicit significant histomorphological, physiological, biochemical or molecular alterations in mouse livers. At the same time, administration of dGTE at MED comparable to those consumed by humans resulted in significant modulation of gut microflora, with increases in Akkermansia sp. being most pronounced. Results of this study demonstrate that administration of relevant-to-human-consumption MED of dGTE to non-fasting mice does not lead to hepatotoxicity. Furthermore, dGTE administered to lean mice, caused changes in gut microflora comparable to those observed in obese mice. This study provides further insight into the previously reported weight management properties of dGTE; however, future studies are needed to fully evaluate and understand this effect.

Impact of obesity on the toxicity of a multi-ingredient dietary supplement, OxyELITE Pro™ (New Formula), using the novel NZO/HILtJ obese mouse model: Physiological and mechanistic assessments.

Herbal dietary supplement (HDS)-induced hepato- and cardiotoxicity is an emerging clinical problem. In this study, we investigated the liver and heart toxicity of HDS OxyELITE-PRO™ New Formula (OEP-NF), a dietary supplement marketed for weight loss and performance enhancement that was recently withdrawn from the market. Using a novel NZO/HlLtJ obese mouse model, we demonstrated that administration of clinically relevant mouse equivalent doses (MED) of OEP-NF produced cardio- and hepatotoxic risks following both short- and long-term administration schedules. Specifically, gavaging female NZO/HlLtJ with up to 2X MED of OEP-NF resulted in 40% mortality within two weeks. Feeding mice with either 1X or 3X MED of OEP-NF for eight weeks, while not exhibiting significant effects on body weights, significantly altered hepatic gene expression, increased the number of apoptotic and mast cells in the heart and affected cardiac function. The degree of toxicity in NZO/HlLtJ mice was higher than that observed previously in non-obese CD-1 and B6C3F1 strains, suggesting that an overweight/obese condition can sensitize mice to OEP-NF. Adverse health effects linked to OEP-NF, together with a number of other hepato- and cardiotoxicity cases associated with HDS ingestion, argue strongly for introduction of quality standards and pre-marketing safety assessments for multi-ingredient HDS.

Safety assessment of the dietary supplement OxyELITE™ Pro (New Formula) in inbred and outbred mouse strains.

Herbal dietary supplements have gained wide acceptance as alternatives to conventional therapeutic agents despite concerns regarding their efficacy and safety. In 2013, a spate of severe liver injuries across the United States was linked to the dietary supplement OxyELITE Pro-New Formula (OEP-NF), a multi-ingredient product marketed for weight loss and exercise performance enhancement. The principal goal of this study was to assess the hepatotoxic potential of OEP-NF in outbred and inbred mouse models. In an acute toxicity study, significant mortality was observed after administering 10X and 3X mouse-equivalent doses (MED) of OEP-NF, respectively. Increases in liver/body weight ratio, ALT and AST were observed in female B6C3F1 mice after gavaging 2X and 1.5X MED of OEP-NF. Similar findings were observed in a 90-day feeding study. These alterations were paralleled by altered expression of gene- and microRNA-signatures of hepatotoxicity, including Cd36, Nqo1, Aldoa, Txnrd1, Scd1 and Ccng1, as well as miR-192, miR-193a and miR-125b and were most pronounced in female B6C3F1 mice. Body weight loss, observed at week 1, was followed by weight gain throughout the feeding studies. These findings bolster safety and efficacy concerns for OEP-NF, and argue strongly for implementation of pre-market toxicity studies within the dietary supplement industry.

Trace element status and zinc homeostasis differ in breast and formula-fed piglets.

Differences in trace element composition and bioavailability between breast milk and infant formulas may affect metal homeostasis in neonates. However, there is a paucity of controlled studies in this area. Here, piglets were fed soy infant formula (soy), cow's milk formula (milk), or were allowed to suckle from the sow from PND2 to PND21. Serum iron concentrations were higher in formula-fed compared to breastfed piglets (P < 0.05). Serum zinc values were higher in milk compared to breastfed or soy groups (P < 0.05). Zinc transporter Zip4 mRNA was elevated in small intestine of the soy compared to breastfed group (P < 0.05). Transporter Znt1 mRNA was greater in small intestine of both formula-fed groups and in liver of the milk compared to the breastfed group (P < 0.05). Metallothionein Mt1 mRNA expression was higher in small intestine and liver of milk compared to breastfed and soy groups (P < 0.05). In liver, metallothionein protein levels and protein bound zinc were also highly elevated in the milk compared to other groups (P < 0.05). mRNA encoding the hepatic zinc-regulated gene Gclc was higher in the milk than soy group (P < 0.05). ChIP assay revealed increased binding of the zinc-regulated transcription factor MTF1 to the promoters of hepatic Mt3 and Gclc genes in the milk compared to the soy group. These data provide evidence that trace element status differs in breastfed, milk-fed, and soy-fed piglets and that despite similar levels of dietary supplementation, allows strong causal inference that significant differences in serum zinc after cow's milk formula compared to soy formula consumption result in compensatory changes in expression of zinc transporters, binding proteins, and zinc-regulated genes.

Feeding soy protein isolate and treatment with estradiol have different effects on mammary gland morphology and gene expression in weanling male and female rats.

Isoflavones are phytochemical components of soy diets that bind weakly to estrogen receptors (ERs). To study potential estrogen-like actions of soy in the mammary gland during early development, we fed weanling male and female Sprague-Dawley rats a semipurified diet with casein as the sole protein source from postnatal day 21 to 33, the same diet substituting soy protein isolate (SPI) for casein, or the casein diet supplemented with estradiol (E2) at 10 µg/kg/day. In contrast to E2, the SPI diet induced no significant change in mammary morphology. In males, there were 34 genes for which expression was changed ≥2-fold in the SPI group vs. 509 changed significantly by E2, and 8 vs. 174 genes in females. Nearly half of SPI-responsive genes in males were also E2 responsive, including adipogenic genes. Serum insulin was found to be decreased by the SPI diet in males. SPI and E2 both downregulated the expression of ERα (Esr1) in males and females, and ERß (Esr2) only in males. Chromatin immunoprecipitation revealed an increased binding of ERα to the promoter of the progesterone receptor (Pgr) and Esr1 in both SPI- and E2-treated males compared with the casein group but differential recruitment of ERß. ER promoter binding did not correlate with differences in Pgr mRNA expression. This suggests that SPI fails to recruit appropriate co-activators at E2-inducible genes. Our results indicate that SPI behaves like a selective estrogen receptor modulator rather than a weak estrogen in the developing mammary gland.