Scientific Program

Conference Series Ltd invites all the participants across the globe to attend 15th Euro-Global Summit on Toxicology and Applied Pharmacology Golden Tulip Berlin – Hotel Hamburg | Berlin, Germany.

Day 1 :

Keynote Forum

Daniele Zink

Institute of Bioengineering and Nanotechnology, Singapore

Keynote: Accurate high-throughput prediction of human organ-specific toxicities

Time : 09:30-10:10

Conference Series Euro Toxicology 2018 International Conference Keynote Speaker Daniele Zink photo
Biography:

Daniele Zink earned her PhD from the University of Heidelberg, Germany in 1995 and completed her Habilitation (2001) at the Ludwig Maximilian University of Munich. In 2001, she was awarded funding of a Junior Group from the Volkswagen-Foundation, which she led until she moved to the Institute of Bioengineering and Nanotechnology (IBN) in Singapore in 2007. Her work on predictive in vitro models has been awarded by Merck Millipore and the US Society of Toxicology. She has won the prestigious LUSH Prize (2016, Science Category). She holds 10 patents/patent applications; is Editorial Board Member of Scientific Reports; has more than 70 peer reviewed publications and is Co-Founder and Director of the spin-off company Cellbae. 

Abstract:

Evaluating the toxicity of chemicals, drug candidates and other compounds requires predictive methods. There is a steeply increasing demand for alternative methods due to various problems associated with animal experiments and changes in legislation (e.g. animal bans for cosmetics testing). However, many alternative methods are of unknown predictivity, and accepted alternative methods for predicting toxicity for human internal organs are not available. This problem is addressed by our work, which was initially focused on the kidney. Recently, we have developed the first animal-free platforms for the accurate prediction of nephrotoxicity in humans. These technologies have received various international awards, including the Lush Science Prize 2016. Our methods include the only available predictive methods based on human induced pluripotent stem cell-derived renal cells and a predictive high-throughput platform. The high-throughput platform is currently applied in collaboration with the US Environmental Protection Agency to predict the human nephrotoxicity of ToxCast compounds. The test balanced accuracies of our predictive methods range between ~80% - 90%, and these methods also reveal injury mechanisms and compound-induced cellular pathways. Based on a similar methodology we are now developing high-throughput platforms for predicting toxicity for other human organ systems, including liver and vasculature. Furthermore, we are establishing predictive organ-on-chip platforms for efficient repeated dose testing and dose-response assessment.

 

Keynote Forum

Merab G Tsagareli

I Beritashvili Center of Experimental Biomedicine, Georgia

Keynote: NSAIDs attenuate agonist-evoked activation of TRP channels

Time : 10:10-10:50

Conference Series Euro Toxicology 2018 International Conference Keynote Speaker Merab G Tsagareli photo
Biography:

Merab Tsagareli is graduated from Tbilisi State University, Georgia (1977) and completed his PhD from Lomonosov State University of Moscow, Russia (1982) and then postdoctoral studies from Serbsky Research Institute for General and Forensic Psychiatry in Moscow, Russia (1985-1990). He is the Director of the Pain and Analgesia Laboratory at Ivane Beritashvili Center for Experimental Biomedicine in Tbilisi, Georgia. His research focuses on the behavioral studies of TRP channels and analgesic and tolerance effects of NSAIDs in relation with the descending pain modulation system. He has published more than 100 papers in peer-reviewed journals.

 

Abstract:

Transient receptor potential (TRP) cations channels are the largest group of sensory detector proteins expressed in the nerve terminals of many receptors including nociceptors and are activated by temperature and chemicals that elicit hot or cold sensations. Antagonists of these channels are likely promising targets for new analgesic drugs at the peripheral and central levels. Because some non-steroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins and NSAIDs attenuate heat nociception and mechanical allodynia in models of inflammatory and neuropathic pain, we investigated whether three widely used NSAIDs (diclofenac, ketorolac, and xefocam) affect thermal and mechanical hyperalgesia following the activation of TRPA1 and TRPV1 channels. We measured nociceptive thermal paw withdrawal latencies and mechanical thresholds bilaterally at various time points following intraplantar injection of the TRPA1 agonists, cinnamaldehyde (CA) and allyl isothiocyanate (AITC) or the TRPV1 agonist capsaicin, or vehicle. When pretreated with vehicle, intraplantar injection of CA, AITC and capsaicin each resulted in significant decreases in thermal withdrawal latency and mechanical threshold in the ipsilateral hindpaw that did not return to baseline for more than 2 hr. To test effects of NSAIDS either diclofenac, ketorolac or xefocam was pre-injected in the same hindpaw 35 min prior to CA, AITC or capsaicin. Pretreatment with each of the three NSAIDs produced strong antinociceptive and antihyperalgesic effects lasting approximately 60 min. Thus, we show for the first time that local administration of NSAIDs suppresses thermal and mechanical hyperalgesia following TRPA1 or TRPV1 activation.

Acknowledgement: The work was supported partially by the grant from Shota Rustaveli National Science Foundation of Georgia (SRNSF #217076).

 

  • Toxicology | Industrial Toxicology | Occupational Toxicology | Methods for Toxicity Testing | Molecular and Biochemical Toxicology | Product Development Toxicology | Environmental Toxicology | Pharmacology
Location: Sylt Foyer
Speaker

Chair

Alexei G. Basnakian

University of Arkansas for Medical Sciences, USA

Speaker

Co-Chair

Frederic J Deschamps

IMTECA, France

Biography:

Abstract:

The contaminanted aquatic environment may end up into the food chain posing risks to tourists’ health in tourist destination. To assess the health risk for tourists visiting st martine island, the best destination of the tourists, both domestic and foreigner, in Bangladesh, a study has been undertaken to analyze the level of heavy metal contamination from Chromium (Cr), Manganese (Mn),Copper (Cu),Zinc(Zn),Arsenic (As),Cadmium (Cd), Lead (Pb), Mercury (Hg), Iron(Fe),  in  six mostly consumed fish (L. fasciatus, R. kanagurta, H. nigrescens, P. cuneatus, P. annularis and S. rubrum),  and five crustacean species (one shrimp (P. sculptilis) one lobster (P. versicolor) and three crabs (P. sanguinolentus, T. crenata and M. victor) captured at St. Martine Island of Bay of Bengal, Bangladesh . The samples were analyzed for trace metals using Atomic Absorption Spectrometer (AAS) and the concentrations of the metals were interpreted using US EPA health risk model. Muscle and carapace/exoskeleton of shrimp, lobster and crabs were analyzed which containted various concentration of Pb, Hg, As, Cr, Cd, Fe, Cu, Zn and Mn . The hiearchy of the havey metal in marine fishes showed as Fe > Cd > Zn > Pb > Cu > Cr > Mn > Hg.  Concentration of Pb in the species R. kanagurta, H. nigresceus and S. rubrum was above  the Food Safety Guideline (FSG) by Australia-New Zealand and other legislation in most of the marine fish and crustaceans. Furthermore, crabs showed the highest mean heavy metal concentrations than that of shrimp and lobster. The carcinogenic acceptable ranges were observed in three fish species (R. kanagurata, H.nigresceus, and S. rubrum) and one crustacean species (P. sculptilis) samples.

Speaker
Biography:

Dirk Petersohn studied biology and conducted his PhD studies at the University of Cologne, Germany on the topic of tissue specific gene regulation in the human nervous system. After his Postdoc studies on wound healing mechanisms in the team of Professor Doctor Thomas Krieg, he joined Henkel AG & Co KGaA in 1998. Since than he held various positions in research and development and is currently the Director of Henkel’s Department of Biological and Clinical Research. Additionally, he Vice-Chairs at Cosmetics Europe’s Steering Scientific committee and Chairs the Task Force Skin Tolerance that is developing approaches to assess skin sensitization of chemicals without the use of animals.

 

Abstract:

Skin is the site of first contact for many chemicals like topically applied dermo-pharmaceuticals, cosmetics or intra-cutaneous injections used in e.g. aesthetic medicine. Traditional two-dimensional monolayer cultures of skin cells are limited in modelling those exposure scenarios. In contrast, Phenion® Full Thickness skin models mimic native human skin in its histological architecture and in a wide spectrum of physiological and biochemical properties. Primary human keratinocytes form a fully differentiated epidermis. It is connected through basement membrane proteins with the underlying dermis which comprises a unique and stable collagen meshwork populated with fibroblasts of the same donor. These conditions boost the de novo synthesis of extracellular matrix proteins like elastin and collagens. Its biological equivalence with human skin makes the tissue model perfectly suited for efficacy testing of substances, e.g. for the intra-cutaneous injection of new hyaluronic acid formulations which are intended to activate gene and protein expression of extracellular matrix molecules. Additionally, toxicological assessments can reliably be performed, as exemplified hereafter for genotoxicity studies. Primary human skin cells in the Phenion® FT skin model keep their normal cell cycle control and DNA-repair competence while additionally exhibiting the xenobiotic metabolism of skin. Beside others, these features make the skin model an ideal tool to be combined with an established toxicological read-out parameter to develop the 3D Skin Comet assay. Recently, the first phase of an ongoing validation exercise has been finalized showing an excellent predictivity in the five participating, international laboratories. Meanwhile, the 3D Skin Comet assay has successfully been used to support the toxicological safety assessment of three cosmetic ingredients. The complete validation data set is expected to build the basis for a broader regulatory acceptance for product risk assessments where the dermal route of exposure is most relevant.

 

Alexei G Basnakian

University of Arkansas for Medical Sciences, USA

Title: DNase-mediated toxicity in normal and cancer cells

Time : 11:45-12:10

Speaker
Biography:

Alexei G Basnakian received his PhD and DSc Degrees from the Russian Academy of Medical Science, both in the field of DNases. He had Postdoctoral trainings in Molecular Biology at Harvard Medical School (USA), and in cancer research at the National Center for Toxicological Research. He is a Professor in the Department of Pharmacology and Toxicology, and Director of the DNA Damage and Toxicology Core Center at the University of Arkansas for Medical Sciences, USA. He is the author of 85 peer-reviewed papers. His research interests lies in DNases and DNA fragmentation associated with cell injury and cell death.

 

 

Abstract:

Endogenous cellular DNases mediate almost all kinds of toxicity to normal and cancer cells, and DNA fragmentation produced by these enzymes makes cell death irreversible. The principal difference between normal and cancer cells is that normal cells express high levels of DNases, while cancer cells have low expression and activity of DNases. The latter is the part of protective mechanism against injury of cancer cells during their growth in a hostile environment. Our studies showed that expression of two most active and abundant DNases, DNase I and EndoG, is strongly suppressed in breast or prostate cancer cell lines, which make them insensitive to anticancer drugs, such as cisplatin, etoposide, camptothecin, and docetaxel. The decrease of EndoG expression is caused by hypermethylation of EndoG gene, while suppression of DNA methylation activated the gene and made cells susceptible to the chemotherapy drugs. The silencing of EndoG using specific siRNA decreased the chemoresistance of the cells, while overexpression of EndoG increased it. The expression of EndoG in orthotopic prostate PC3 cell xenografts in mice increased sensitivity of the tumors to docetaxel. Off-target toxic effect of anticancer drugs on normal tissues was also mediated by DNases in several models. Administration of cisplatin induced toxicity to kidneys, cyclophosphamide induced alopecia, and administration of cisplatin, vinblastin or doxorubicin were toxic to vascular endothelial cells. Importantly, in all of these cases, genetic knockout of chemical inhibition of endonucleases were protective to normal tissues. Therefore, activation of DNases in cancer tissues while inactivating them in normal tissues should be considered as viable approach in cancer therapy.

 

Speaker
Biography:

Frederic J Deschamps is a Medical Occupational Doctor since 1990. He is a Professor of Medicine since 2002. He manages Department of Occupational Diseases at the University Hospital of Reims. He is the Director of the Department of Occupational Health. His main topic concerns the assessment of diseases in relationship with low level toxic during long periods. He is mainly interested in: occupational health and toxicology.

 

Abstract:

Occupational exposure is a factor closely related to few immune system diseases. An association between occupational exposures of inhaled dust, and auto-immunity, was postulated as early XXe century. Genetic factors exist and affect the development of systemic autoimmune disease in certain individuals. But occupational factors could also play a substantial role. The objective was to explore the potential association between dust and airborne hairdresser's exposure inducing autoimmune diseases, more specially Systemic Lupus Erythematous (SLE).The study investigated the relationship of occupational dust inhalation exposure in hairdressing salons, and onset of SLE. Cases were defined as, those who reported having SLE, with clinical and biological confirmation. Cases were more likely to have been exposed to Hairdressing Dust Inhalation Exposure (HDIE), than other people without the disease. Onset of SLE appears in a close temporal relationship with HDIE. The development of symptoms of fatigue, myalgia, arthralgia, dysesthesias of the hands and feet and skin problems, appeared to be linked to HDIE. Moreover, increase positive autoantibodies have been observed, followed by a decrease, after cessation of HDIE. These biomarkers were dependant of HDIE. SLE may be caused by a large number of chemically unrelated agents under circumstances of exposure, which were prolonged and frequent. Agents implicated in
triggering or accelerating SLE were mercury, iodine, vinyl chloride, and crystalline silica, possibly included in hairdressing products. But, it is difficult to infer correctly the exact agent because hairdressing products are made of many chemicals and mixtures. It is important to underline that dust itself has adjuvant effects. In conclusion, mechanism and epidemiology linking HDIE to development of SLE, are not well known. But these findings support the hypothesis that HDIE is frequently associated with SLE.

Speaker
Biography:

Najah Abi Gerges, PhD, is the Vice President of Research and Development at AnaBios Corporation, USA. He holds a PhD in Cardiac Physiology from Paris XI University, France. Prior to joining AnaBios, he was involved in drug discovery programs at AstraZeneca. With over 17 years in the pharmaceutical industry, he is an innovative Leader, having made substantial contributions to drug approvals (Tagrisso®), research across several areas of cardiac physiology and pharmacology resulting in over 40 published articles, and novel paradigms to advance cardiovascular translational science. He is the Editor for the Journal of Pharmacological and Toxicological Methods, Vice President Elect for Southern California Chapter of the Society of Toxicology. His interest lies in drug discovery and development. 

 

Abstract:

Drug-induced pro-arrhythmia and/or changes in contractility can limit the utility of potential novel therapeutics. Since abnormal ventricular repolarization can cause not only electrical disorders, but also affect the heart’s contractile function, we developed a new model based on adult human primary cardiomyocytes to provide a preclinical tool for the simultaneous prediction of drug-induced inotropic and pro-arrhythmia risks. We recorded fractional sarcomere shortening (SS) using a digital, cell geometry measurement system (IonOptix™) and then record changes in the contractility transients to infer both inotropic (SS) as well as pro-arrhythmia risk (aftercontraction). Validation data were generated with 38 clinically well characterized controls: 23 torsadogenic and 10 non-torsadogenic drugs, and 5 positive inotropes. When the assessment of pro-arrhythmia risk was based on effects observed at 10x of the free effective therapeutic plasma concentration, human cardiomyocyte-based model had excellent assay 96% sensitivity and 100% specificity. Human cardiomyocytes also identified drugs associated with negative and positive inotropic effects. Moreover, positive inotrope-induced changes in contractility parameters illustrated the potential for finger-printing different mechanisms of action. Thus, human cardiomyocytes can simultaneously predict risks associated with pro-arrhythmia and inotropic activity. This approach enables the generation of predictive preclinical human-based cardiotoxicity data and appears to be more predictive than the stem cell-derived cardiomyocyte models.

 

Speaker
Biography:

Jianwei Zhou has his expertise in research on DNA damage and repair, cancer initiation, metastasis and drug resistance. Based on the mechanistic discovery of JWA gene in anticancer and neuroprotection, he has developed anticancer polypeptides and small molecular compounds and these will can be potentially used as neurodegenerative protection and cancer metastasis therapeutic agents.

 

 

Abstract:

Paraquat (PQ), a widely used environmental toxin in agriculture, contributes to the onset and progression of Parkinson’s disease (PD) by damaging the neurons. The JWA gene, also known as ARL6IP5, exhibits the protective effect on dopamine (DA) neuron degeneration. In our study, neuronal and astrocytic JWA knockout (JWA-nKO and JWA CKO) mice were used to exposure of PQ, two neuron cell lines (HT-22, SH-SY5Y) and primary astrocytes were also subjected to PQ treatment. The results showed that PQ administration triggered the upregulation of JWA. Elevated expressions of JWA rescued the redundant abundance of reactive oxygen species (ROS) while increased the levels of glutathione (GSH) and glutathione peroxidase (GPx) under PQ exposure. Astrocytic JWA deficiency repressed expression of excitatory amino acid transporter 2 (GLT-1) and glutamate uptake both in vivo and in vitro. The further mechanistic data indicated that the protective role of JWA in dopaminergic neurons were mainly through anti-oxidative stress induced DNA damage by regulating MEK/PI3K-Nrf2 axis; however it were mediated by MEK/PI3K-GLT1 signaling in astrocytes, through maintaining homeostasis of intracellular excitatory glutamate; and this was confirmed in MPTP/p-induced PD mice model. Taken together, our findings provide novel insights for both neuronal and astrocytic JWA functions in the pathogenesis of neurotoxin mouse models of Parkinson's disease.

 

 

Speaker
Biography:

Eva Novotna has her expertise in the preparation and purification of recombinant enzymes. She studies interactions of potential inhibitors with carbonyl reducing enzymes using human recombinant enzymes and cancer cell lines. Her research interest include: carbonyl reducing enzymes, inhibitors and cancer drug resistance.

 

Abstract:

Dinaciclib (MK-7965, SCH727965) is a cyclin-dependent kinase inhibitor. Dinaciclib recently enters Phase III clinical trial for the treatment of leukaemia. The results of the study shows its promising anti-leukemia activity and a tolerability. Dinaciclib causes apoptosis to several cancer cell lines, including those that are resistant to anthracyclines. The reductive metabolism of anthracyclines to their secondary C13-hydroxy metabolites is one of the main mechanisms leading to cancer resistance. To date, this kind of metabolism has been associated with members of the short-chain dehydrogenase/reductase (SDR) and of aldo-keto reductase superfamilies (AKR). In our study, dinaciclib was identified as an effective inhibitor of aldo-keto reductase 1C3 (AKR1C3), the enzyme that is overexpressed in many cancer types and its increased metabolism contributes to the pharmacokinetic resistance to antracyclines in tumor tissues. In our study, dinaciclib inhibited AKR1C3 in the experiments with purified recombinant enzyme (IC50=220 nM, Ki=170 nM) and was equally active at the cellular level (IC50=235 nM). Molecular docking studies predicted that dinaciclib occupies a part of the cofactor binding site, which is consistent with the noncompetitive mechanism of inhibition determined in our in vitro experiments. Furthermore, we demonstrated that pretreatment with dinaciclib significantly sensitized AKR1C3-overexpressing anthracycline-resistant cancer cells to daunorubicin. Our results indicate that dinaciclib may potentially increase the therapeutic efficacy of anthracyclines, prevent anthracycline resistance and minimize their adverse effects.

 

Speaker
Biography:

Tim Meyer has his expertise in tissue engineering  of human myocardium and the automation of tissue generation and analysis.  Coming from a multidisciplinary, engineering background his focus is on methods development, 3D printing, video analysis and laboratory automation

Abstract:

We employ engineered human myocardium (EHM) as patient-centric model for preclinical safety and efficacy screens, replacing animal research.

Based on the spontaneous and stimulated auxotonic contraction exhibited by EHM we developed a 48 well screening plate for chronic and acute screens of potentially cardio-active compounds. EHM are cast and suspended on fluorescent rubber poles generating a defined restoring force, contractions are recorded at high spatial (20µm) and temporal (20ms) resolution and analyzed in real time using special purpose image recognition hardware. Based on contraction frequency and amplitude we determine, besides others, inotropic, chronotropic, lusotropic and arrhythmogenic responses to treatment.

Our video-optical measurements are sterile and non-perturbing allowing successive measurements over weeks and months to follow tissue maturation and chronic effects. Continuous electrical stimulation may be applied for improved maturation and as a tachycardia model. Poles with increasing stiffness simulate pressure overload. The screening platform is validated against reference compounds, comparison to our classical isometric organ bath measurements and clinical data.

 

Liu Zheng Tao

Chinese Research Academy of Environmental Sciences, China

Title: Environmental pollution and water quality criteria of perfluorinated chemicals in China
Speaker
Biography:

Liu Zhengtao has his expertise in ecotoxicology, environmental criteria and risk assessment in China.

 

Abstract:

Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are emerging persistent organic pollutants, the global eco-system of pollution caused by them have become a fact. In this study, the use and the physico-chemical properties of perfluorinated compounds (PFCs) were summarized. The pollution level of PFOS and PFOA in environment media (water; atmospheric; soil and the deposition) and organisms in China were analyzed. Moreover, toxicity data of aquatic and terrestrial species were screened and selected, and the water quality criteria of PFOS and PFOA pollutants were developed based on Chinese native species. Thereafter, ecological risk assessment of PFCs was carried out based on the environmental concentration level and the water quality criteria of PFCs in China.

 

 

Speaker
Biography:

Wang Xiaonan has his expertise in ecotoxicology, environmental criteria and risk assessment in China.    

 

Abstract:

Systematic study about water quality criteria is being carried out in China, but study of soil environmental criteria is comparatively insufficient. In this study, germination and root growth of 8 terrestrial plants (Triticum aestivum, Lactuca sativa, Cucumis sativus, Zea mays, Brassica pekinensis, Glycine max, Allium tuberosum and Solanum lycopersicum) and growth inhibition of one terrestrial animal (Achatina fulica) were used to determine the chronic ecotoxicological effects of chromium (Ⅵ) using the agricultural moisture soil of Baoding. In addition, with the native toxicity data selected, the HC5 (hazardous concentration of the 5% species) and the ecological protected soil environmental criteria of chromium (Ⅵ) in Baoding moisture soil were calculated using the log-normal species sensitivity distribution (SSD) method. Results showed that NOEC (no observed effect concentration) values for the growth of the terrestrial plants T. aestivum, L. sativa, C. sativus, Z. mays, B. pekinensis, G. max, A. tuberosum, S. lycopersicum, and the terrestrial invertebrate snail A. fulica were 19.0, 21.0, 28.0, 32.0, 28.0, 32.0, 32.0, 12.0 and 20.0 mg/kg, respectively. The comparison of species toxicity data that were tested in the same conditions showed that terrestrial plants S. lycopersicum was the most sensitive species to chromium (Ⅵ), T. aestivum and L. sativa had the same sensitivities to chromium (Ⅵ) exposure, whereas, plants C. sativus, Z. mays, B. pekinensis, G. max and A. tuberosum had the same sensitivities to chromium (Ⅵ) exposure. Finally, the HC5 value of chromium (Ⅵ) in the moisture soil of Baoding was calculated to be 7.66 (4.12<CI<11.34) mg/kg using the log-normal SSD method, and the ecologically protected soil environmental criteria of chromium (Ⅵ) was 1.53~7.66 mg/kg. With the investigation of this work, we expect that it could provide useful information in the study of soil environmental criteria in China.

 

Speaker
Biography:

Yu Jie has his expertise in Environmental Toxicology. His most recent researches focus on the impact of environmental endocrine disruptors on endocrine metabolism disorder, neurotic depression and its mechanisms.

 

 

Abstract:

Exposure to environmental endocrine disruptors (EEDs) contributes to the pathogenesis of many metabolic disorders. Here, we have analyzed the effect of the EED-nonylphenol (NP) in the promotion of non-alcoholic fatty liver disease (NAFLD) in rats fed high sucrose-high fat diet (HSHFD). Fifty Sprague-Dawley rats were divided into five groups: controls fed a normal diet (C-ND); HSHFD-fed controls (C-HSHFD); and rats fed a HSHFD combined with NP at doses of 0.02 μg/kg/day (NP-L-HSHFD), 0.2 μg/kg/day (NP-M-HSHFD), and 2 μg/kg/day (NP-H-HSHFD). Subchronic exposure to NP coupled with HSHFD increased daily water and food intake (p<0.05), hepatic echogenicity and oblique liver diameter (p<0.05), and plasma levels of alanine aminotransferase, aspartate aminotransferase, total cholesterol, triglycerides, and low density lipoprotein cholesterol (p<0.05). Combined exposure to NP and HSHFD induced macrovesicular steatosis with dilation and congestion of the central vein, liver inflammatory cell infiltration, and expression of genes regulating lipid metabolism, SREBP-1C, FAS, and Ucp2. These results demonstrate that NP aggravates NAFLD in HSHFD-treated rats by up-regulating lipogenic genes, and that HSHFD increases the toxic effects of NP. Thus subchronic NP exposure may lead to NAFLD, especially when combined with a high-sucrose/high-fat diet.