Researcher views cell images on computer in lab
Our current research activities are devoted to understanding the safety and efficacy of dietary supplements, as well as the development of new natural products.

Below is a list of current and recent projects.

Research Projects

Molecular mechanisms of OxyELITE Pro-New Formula toxicity

Project Leads: Drs. Bill Gurley, Igor Koturbash, and Marjan Boerma
Study Support: U. S. Department of Justice (contract number: 6L-CIV02-0850)

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 lean and obese 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.

Furthermore, using a novel NZO/HlLtJ obese mouse model, we demonstrated that administration of clinically relevant MED of OEP-NF produced cardio- and hepatotoxic risks following both short- and long-term administration schedules. Specifically, gavaging female NZO/HlLtJ mice 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 cells and the number of mast cells in the heart and reduced 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.

Publications in peer-reviewed journals on this subject:

Miousse IR, Skinner CM, Lin H, Ewing LE, Kosanke SD, Williams DK, Avula B, Khan IA, ElSohly MA, Gurley BJ, Koturbash I. Safety assessment of the dietary supplement OxyELITE™ Pro (New Formula) in inbred and outbred mouse strains. Food Chem Toxicol. 2017 Nov;109(Pt 1):194-209. doi: 0.1016/j.fct.2017.08.025. Epub 2017 Aug 24. PubMed PMID: 28843594. Read about this study

Skinner CM, Miousse IR, Ewing LE, Sridharan V, Cao M, Lin H, Williams DK, Avula B, Haider S, Chittiboyina AG, Khan IA, ElSohly MA, Boerma M, Gurley BJ, Koturbash I. 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. Food Chem Toxicol. 2018 Dec;122:21-32. doi: 10.1016/j.fct.2018.09.067. Epub 2018 Sep 30. PubMed PMID:30282009; PubMed Central PMCID: PMC6219625. Read about this study

Green Tea Extract (GTE) Studies

Project Leads: Drs. Igor Koturbash and Bill Gurley

The goals of this study are to investigate the hepatotoxic potential and effects on the gut microbiome of decaffeinated green tea extract (dGTE) in lean B6C3F1 mice. Gavaging within the range of 1X – 10X mouse equivalent doses (MED) of dGTE for up to two weeks did not elicit significant histomorphological, physiological, biochemical or molecular alterations in the mouse livers. At the same time, MED of dGTE relevant to human consumption, resulted in modulation of the gut microbiome with the most pronounced increase in Akkermansia sp.

The 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 causes changes in their gut microbiome comparable to those observed in obese mice. This study provides further insights into the previously reported weight management properties of GTE; however, future studies are clearly needed to fully evaluate and understand this effect. These studies are underway in our laboratories.

Publications in peer-reviewed journals on this subject:

Gurley BJ, Miousse IR, Nookaew I, Ewing LE, Skinner CM, Jenjaroenpun P, Wongsurawat T, Kennon-McGill S, Avula B, Bae JY, McGill MR, Ussery D, Khan IA, Koturbash I. Decaffeinated Green Tea Extract Does Not Elicit Hepatotoxic Effects

and Modulates the Gut Microbiome in Lean B6C3F₁ Mice. Nutrients. 2019 Apr 3;11(4). pii: E776. doi: 10.3390/nu11040776. PubMed PMID: 30987244. Read more about this study

Hepatotoxicity of Cannabidiol

Project Leads: Drs. Igor Koturbash and Bill Gurley

Cannabidiol (CBD) is a non-psychotropic ingredient of Cannabis sativa. In recent years, its utilization progressively increased due to the expanded availability of CBD to the general public together with aggressive marketing. Emerging evidence, however, indicates that CBD poses a risk for hepatotoxicity. The goal of this study was to investigate the hepatotoxic potential of CBD delivered with sesame oil to 8-week-old male B6C3F1 mice. Animals were gavaged with single allometrically-scaled mouse equivalent doses (MED) of either 0, 20, 60 or 200 mg/kg of CBD (acute toxicity, 24 h) or with daily MED of 0, 5, 15 or 50 mg/kg for 10 days. In the acute study, besides increases in the liver-to-body weight (LBW) ratio, gavaging mice with 200 mg/kg of CBD resulted in increased plasma concentrations of ALT and AST and spiking (~20-fold) levels of total bilirubin. In a two-week study, 50% of mice gavaged with 50 mg/kg developed severe toxicity manifested as tremors and subsequent lethargy starting after the second dose of CBD. Similar to findings in the acute phase, 50 mg/kg CBD increased LBW ratios, ALT, AST and total bilirubin. Hepatotoxicity gene expression arrays revealed that more than 50 genes were differentially regulated following CBD administration. Several dysregulated genes were associated with oxidative stress responses, lipid metabolism and a number of enzymes responsible for drug and ethanol biotransformation. In conclusion, mice gavaged with clinically-relevant MED of CBD exhibited clear signs of liver injury. Involvement of numerous pathways associated with lipid and xenobiotic metabolism raises serious concerns about potential drug interactions as well as the safety of CBD.

Further studies on CBD/drug interactions are underway in our laboratories.

Publications in peer-reviewed journals on this subject:

Ewing LE, Skinner CM, Quick CM, Kennon-McGill S, McGill MR, Walker LA, ElSohly MA, Gurley BJ, Koturbash I. Hepatotoxicity of a Cannabidiol-Rich Cannabis Extract in the Mouse Model. Molecules. 2019 Apr 30;24(9). pii: E1694. doi:

10.3390/molecules24091694. PubMed PMID: 31052254. Read more about this study

Phosphatidic acid as a treatment for acute liver injury and liver failure

Project Lead: Dr. Mitchell McGill
Study Support: American Association for the Study of Liver Diseases Foundation

Acute liver failure (ALF) is a rare but often fatal condition. Mortality varies by etiology, but can be as high as 80%. The major cause of ALF in the United States and several other countries is overdose of the analgesic acetaminophen (APAP). Although there is a safe and effective antidote for APAP toxicity (N-acetylcystegine), it loses efficacy >16 h after overdose. After that, the only life-extending treatment is a liver transplant. However, there is a shortage of transplantable organs. Furthermore, transplant recipients typically require immunosuppressants to avoid organ rejection, and they face myriad other complications. Our laboratory recently discovered that the glycerophospholipid (GPL) phosphatidic acid (PA) is critical for liver repair and regeneration after APAP overdose. PA is the major precursor for synthesis of new cell membrane GPLs, as well as triacylglycerols. It is also a major signaling mediator in cells. We have demonstrated that reducing PA content in hepatocytes using various approaches decreases hepatocyte proliferation and pro-proliferative signaling. We recently observed that treatment with exogenous PA can also protect against APAP-induced liver injury. Importantly, PA is already available over-the-counter as a dietary supplement, so we are now testing supplemental PA as a readily-available treatment for ALF.

Genetic variability in DHFR and folic acid supplementation as novel risk factors for aggressive prostate cancer

Project Lead: Dr. Joseph Su
Study Support: National Cancer Institute (grant number: R15CA231510)

Folate and its synthetic form, folic acid, is a water-soluble B vitamin which is involved in DNA synthesis and repair, and in regulation of gene expression through DNA methylation as a methyl donor. Despite the confirmed beneficial effect of folate/folic acid supplementation on the prevention of neural tube defect, concerns have been raised recently that excessively high intakes may promote carcinogenesis or cancer progression. In 2015, the NIH National Toxicology Program (NTP) and Office of Dietary Supplements (ODS) Cancer Subpanel concluded, “There is consistent enough suggestion in human studies of an adverse effect on cancer growth from supplemental folic acid to justify further research.” Circulating unmetabolized folic acid (cUMFA) is a by-product of high intake of folic acid and has been associated with reduction of cytotoxicity of natural killer cells, which may be a mechanism by which high levels of folic acid promote carcinogenesis. Study has shown that participants with dihydrofolate reductase (DHFR) 19bpdel/del, which is more prevalent among African Americans (AAs), were at increased risk of having elevated levels of cUMFA when daily folic acid intake exceeded 500 micrograms. At the same time, AAs are diagnosed with aggressive prostate cancer more often and have more than twice the prostate cancer mortality rates as European Americans (EAs). Therefore, we hypothesized that excess folic acid intake, as measured by both diet/supplement intake and plasma cUMFA, will be associated with increased odds of highly aggressive prostate cancer, and that this effect will be modified by the DHFR polymorphism. This study capitalizes on a large, previously-conducted, well-characterized, case-only study of an approximately equal number of AAs and EAs to examine folate/folic acid intake, biomarkers, genetic polymorphisms and racial differences in prostate cancer aggressiveness. The finding of this study, if our hypothesis is confirmed, will shed light into concerns about the safety of folic acid supplements and inform dietary guidelines for cancer prevention.

Development of γ-tocotrienol as radiation countermeasure for astronauts

Project Lead: Dr. Marjan Boerma
Study support: National Space Biology Research Institute (grant number: RE03701 through NCC 9-58), NASA (grant numbers: 80NSSC17K0425 and 80NSSC19K0437)

On future missions into deep space, astronaut crews will be exposed to ionizing radiation that increases the risk of developing cancer and degenerative effects in organ systems such as the central nervous system and the heart. Administration of a safe dietary supplement with protective properties against adverse effects of radiation before and during the mission is an attractive option to reduce these risks. γ-Tocotrienol is a natural form of vitamin E that differs from the most common form of vitamin E, α-tocopherol, in its unsaturated farnesyl side chain. Tocotrienols have received attention in four areas of tissue protection that argue for their value as a promising countermeasure against space radiation: 1) Tocotrienols are among the strongest radiation protectors of all natural compounds tested to date; 2) Tocotrienols have beneficial biological properties that promote cardiovascular health. Both δ- and γ-tocotrienol are the only known forms of vitamin E that inhibit the cholesterol biosynthesis pathway; 3) Tocotrienols protect against neurodegeneration in animal models and human subjects and 4) Several studies show that tocotrienols have cancer preventive properties. Efforts are well underway to develop γ-tocotrienol as a countermeasure against the acute radiation syndrome caused by accidental radiation exposure on Earth. Moreover, several current clinical trials are recruiting patients to test tocotrienols in the treatment of cancer, liver disease and brain injury from stroke. Therefore, we hypothesize that γ-tocotrienol protects against carcinogenesis and adverse effects in the central nervous system and cardiovascular system from exposure to radiation in deep space. We use animal models of exposure to space-like radiation to test the effects of γ-tocotrienol on tumor formation, cognitive and cardiac function and tissue structure. First results of these studies indicate that γ-tocotrienol mitigates radiation-induced increases in immune cell markers in the heart.

The goal of this project was to characterize the risks of and identify interventions in cardiovascular injury from exposure to space radiation.

Dietary modulation of normal and cancerous tissue response to ionizing radiation

Project Lead: Dr. Igor Koturbash
Study Support: National Institute of General Medical Sciences (grant number: 1P20GM109005)

Methionine is an essential amino acid needed for a variety of processes in living organisms. Ionizing radiation depletes the tissue methionine concentrations and leads to the loss of DNA methylation and decreased synthesis of glutathione. Therefore, methionine dietary supplementation (MDS) was proposed to improve the response to total body irradiation (TBI). Strikingly, our studies indicated that MDS potentiated acute radiation toxicity, resulting in earlier lethality at a sub-lethal dose of 8.5 Gy compared to mice fed methionine-adequate diet (MAD). Hind-limb protection during exposure to 8.5 Gy of TBI resulted in 50% mortality at days 7-9 post irradiation in mice on MDS, while all mice fed MAD survived 30 days after TBI. These findings suggest the prevalence of gastrointestinal syndrome in MDS-induced acute radiation toxicity. MDS alone has led to substantial alterations in the one-carbon metabolism in both proximal jejunum and liver of mice, loss of retrotransposon LINE-1 DNA methylation, and negatively regulated the expression of tight junction genes in the proximal jejunum. Furthermore, MDS caused significant shifts in the intestinal microbiota profiles. At the same time, administration of methionine-deficient diets (MDD) substantially delayed the onset of acute radiation syndrome after exposure to lethal doses of TBI.

On the other hand, methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation (MDD) at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In the present study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and likely most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Publications in peer-reviewed journals on this subject:

Koturbash I, Griffin RJ. Harnessing epigenetics and metabolism to modulate tissue response to radiotherapy. Int J Radiat Biol. 2019 Mar 11:1-3. doi: 10.1080/09553002.2019.1587268. [Epub ahead of print] PubMed PMID: 30856046. Read about this study

Miousse IR, Tobacyk J, Quick CM, Jamshidi-Parsian A, Skinner CM, Kore R, Melnyk SB, Kutanzi KR, Xia F, Griffin RJ, Koturbash I. Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors. Carcinogenesis. 2018 Sep 21;39(9):1117-1126. doi: 10.1093/carcin/bgy085. PubMed PMID: 29939201; PubMed Central PMCID: PMC6148987. Read about this study

Koturbash I. 2017 Michael Fry Award Lecture When DNA is Actually Not a Target: Radiation Epigenetics as a Tool to Understand and Control Cellular Response to Ionizing Radiation. Radiat Res. 2018 Jul;190(1):5-11. doi: 10.1667/RR15027.1. Epub 2018 Apr 26. PubMed PMID: 29697303; PubMed Central PMCID: PMC6036898. Read about this study

Miousse IR, Pathak R, Garg S, Skinner CM, Melnyk S, Pavliv O, Hendrickson H, Landes RD, Lumen A, Tackett AJ, Deutz NEP, Hauer-Jensen M, Koturbash I. Short-term dietary methionine supplementation affects one-carbon metabolism and DNA methylation in the mouse gut and leads to altered microbiome profiles, barrier function, gene expression and histomorphology. Genes Nutr. 2017 Sep 6;12:22. doi: 10.1186/s12263-017-0576-0. eCollection 2017. PubMed PMID: 28904640; PubMed Central PMCID: PMC5588631. Read about this study

Miousse IR, Tobacyk J, Melnyk S, James SJ, Cheema AK, Boerma M, Hauer-Jensen M, Koturbash I. One-carbon metabolism and ionizing radiation: a multifaceted interaction. Biomol Concepts. 2017 May 24;8(2):83-92. doi: 10.1515/bmc-2017-0003. Review. PubMed PMID: 28574375. Read about this study