Maternal high fat diet is associated with decreased plasma n-3 fatty acids and fetal hepatic apoptosis in nonhuman primates

PLoS ONE

Volumn 6, Issue 2, 2011, Pages

  Grant, Wilmon F ^a,b   Gillingham, Melanie B ^a   Batra, Ayesha K ^a   Fewkes, Natasha M ^b   Comstock, Sarah M ^c   Takahashi, Diana ^c   Braun, Theodore P ^b   Grove, Kevin L ^c   Friedman, Jacob E ^d   Marks, Daniel L ^a,b  

^a OREGON HEALTH AND SCIENCE UNIVERSITY   (United States)

^b OREGON HEALTH AND SCIENCE UNIVERSITY   (United States)

^c OREGON HEALTH AND SCIENCE UNIVERSITY   (United States)

^d Children's Hospital Colorado   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARACHIDONIC ACID; DOCOSAHEXAENOIC ACID; ESSENTIAL FATTY ACID; ICOSAPENTAENOIC ACID; LINOLEIC ACID; MONOUNSATURATED FATTY ACID; MYRISTIC ACID; MYRISTOLEIC ACID; OLEIC ACID; OMEGA 3 FATTY ACID; OMEGA 6 FATTY ACID; PALMITIC ACID; PALMITOLEIC ACID; POLYUNSATURATED FATTY ACID; STEARIC ACID; UNCLASSIFIED DRUG;

ANIMAL EXPERIMENT; ANIMAL MODEL; ANIMAL TISSUE; ARTICLE; BREAST MILK; CONTROLLED STUDY; FATTY ACID BLOOD LEVEL; FEMALE; FETUS; FETUS DEVELOPMENT; FETUS LIVER; LIPID COMPOSITION; LIPID DIET; LIVER DEVELOPMENT; LIVER NECROSIS; MATERNAL NUTRITION; NONHUMAN; NUTRITIONAL ASSESSMENT; OBESITY; PERINATAL PERIOD; PHENOTYPE; PREGNANCY; PRIMATE; PROTEIN BLOOD LEVEL; ANIMAL; APOPTOSIS; ATHEROGENIC DIET; BLOOD; CHEMISTRY; DRUG EFFECT; EVALUATION; FAT INTAKE; FETUS BLOOD; HUMAN; LIVER; METABOLISM; PATHOLOGY; PHYSIOLOGY; PRENATAL DEVELOPMENT; PRENATAL EXPOSURE; RANDOMIZATION;

PRIMATES;

ANIMALS; APOPTOSIS; DIET, ATHEROGENIC; DIETARY FATS; FATTY ACIDS, OMEGA-3; FEMALE; FETAL BLOOD; FETUS; HUMANS; LIVER; MATERNAL NUTRITIONAL PHYSIOLOGICAL PHENOMENA; PREGNANCY; PRENATAL EXPOSURE DELAYED EFFECTS; PRIMATES; RANDOM ALLOCATION;

EID: 79952140771     PISSN: None     EISSN: 19326203     Source Type: Journal    
DOI: 10.1371/journal.pone.0017261     Document Type: Article

References (104)

1. Mokdad AH, Ford ES, Bowman BA, Dietz WH, Vinicor F, et al. (2003) Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001. Jama 289: 76-79.
2. Mokdad AH, Serdula MK, Dietz WH, Bowman BA, Marks JS, et al. (1999) The spread of the obesity epidemic in the United States, 1991-1998. Jama 282: 1519-1522.
3. Vahratian A, (2008) Prevalence of Overweight and Obesity Among Women of Childbearing Age: Results from the 2002 National Survey of Family Growth. Matern Child Health J.
4. Hedley AA, Ogden CL, Johnson CL, Carroll MD, Curtin LR, et al. (2004) Prevalence of overweight and obesity among US children, adolescents, and adults, 1999-2002. Jama 291: 2847-2850.
5. Ogden CL, Carroll MD, Curtin LR, McDowell MA, Tabak CJ, et al. (2006) Prevalence of overweight and obesity in the United States, 1999-2004. Jama 295: 1549-1555.
6. Pinhas-Hamiel O, Zeitler P, (2005) The global spread of type 2 diabetes mellitus in children and adolescents. J Pediatr 146: 693-700.
7. Pinhas-Hamiel O, Zeitler P, (2007) Acute and chronic complications of type 2 diabetes mellitus in children and adolescents. Lancet 369: 1823-1831.
8. Bispham J, Gardner DS, Gnanalingham MG, Stephenson T, Symonds ME, et al. (2005) Maternal nutritional programming of fetal adipose tissue development: differential effects on messenger ribonucleic acid abundance for uncoupling proteins and peroxisome proliferator-activated and prolactin receptors. Endocrinology 146: 3943-3949.
9. Budge H, Gnanalingham MG, Gardner DS, Mostyn A, Stephenson T, et al. (2005) Maternal nutritional programming of fetal adipose tissue development: long-term consequences for later obesity. Birth Defects Res C Embryo Today 75: 193-199.
10. Oken E, Taveras EM, Kleinman KP, Rich-Edwards JW, Gillman MW, (2007) Gestational weight gain and child adiposity at age 3 years. Am J Obstet Gynecol 196: 322 e321-328.
11. Painter RC, de Rooij SR, Bossuyt PM, de Groot E, Stok WJ, et al. (2007) Maternal nutrition during gestation and carotid arterial compliance in the adult offspring: the Dutch famine birth cohort. J Hypertens 25: 533-540.
12. Stein AD, Ravelli AC, Lumey LH, (1995) Famine, third-trimester pregnancy weight gain, and intrauterine growth: the Dutch Famine Birth Cohort Study. Hum Biol 67: 135-150.
13. Stein AD, Zybert PA, van de Bor M, Lumey LH, (2004) Intrauterine famine exposure and body proportions at birth: the Dutch Hunger Winter. Int J Epidemiol 33: 831-836.
14. Stein Z, Susser M, (1975) The Dutch famine, 1944-1945, and the reproductive process. II. Interrelations of caloric rations and six indices at birth. Pediatr Res 9: 76-83.
15. Stocker CJ, Arch JR, Cawthorne MA, (2005) Fetal origins of insulin resistance and obesity. Proc Nutr Soc 64: 143-151.
16. Boney CM, Verma A, Tucker R, Vohr BR, (2005) Metabolic syndrome in childhood: association with birth weight, maternal obesity, and gestational diabetes mellitus. Pediatrics 115: e290-296.
17. Dabelea D, Hanson RL, Lindsay RS, Pettitt DJ, Imperatore G, et al. (2000) Intrauterine exposure to diabetes conveys risks for type 2 diabetes and obesity: a study of discordant sibships. Diabetes 49: 2208-2211.
18. Dabelea D, Pettitt DJ, Hanson RL, Imperatore G, Bennett PH, et al. (1999) Birth weight, type 2 diabetes, and insulin resistance in Pima Indian children and young adults. Diabetes Care 22: 944-950.
19. Garcia Carrapato MR, (2003) The offspring of gestational diabetes. J Perinat Med 31: 5-11.
20. Plagemann A, Harder T, Kohlhoff R, Rohde W, Dorner G, (1997) Overweight and obesity in infants of mothers with long-term insulin-dependent diabetes or gestational diabetes. Int J Obes Relat Metab Disord 21: 451-456.
21. Plagemann A, Harder T, Kohlhoff R, Rohde W, Dorner G, (1997) Glucose tolerance and insulin secretion in children of mothers with pregestational IDDM or gestational diabetes. Diabetologia 40: 1094-1100.
22. Silverman BL, Landsberg L, Metzger BE, (1993) Fetal hyperinsulinism in offspring of diabetic mothers. Association with the subsequent development of childhood obesity. Ann N Y Acad Sci 699: 36-45.
23. Van Assche FA, Holemans K, Aerts L, (2001) Long-term consequences for offspring of diabetes during pregnancy. Br Med Bull 60: 173-182.
24. Whitaker RC, (2004) Predicting preschooler obesity at birth: the role of maternal obesity in early pregnancy. Pediatrics 114: e29-36.
25. Yogev Y, Langer O, (2008) Pregnancy outcome in obese and morbidly obese gestational diabetic women. Eur J Obstet Gynecol Reprod Biol 137: 21-26.
26. Anguita RM, Sigulem DM, Sawaya AL, (1993) Intrauterine food restriction is associated with obesity in young rats. J Nutr 123: 1421-1428.
27. Chang GQ, Gaysinskaya V, Karatayev O, Leibowitz SF, (2008) Maternal high-fat diet and fetal programming: increased proliferation of hypothalamic peptide-producing neurons that increase risk for overeating and obesity. J Neurosci 28: 12107-12119.
28. Davidowa H, Heidel E, Plagemann A, (2002) Differential involvement of dopamine D1 and D2 receptors and inhibition by dopamine of hypothalamic VMN neurons in early postnatally overfed juvenile rats. Nutr Neurosci 5: 27-36.
29. Davidowa H, Li Y, Plagemann A, (2002) Hypothalamic ventromedial and arcuate neurons of normal and postnatally overnourished rats differ in their responses to melanin-concentrating hormone. Regul Pept 108: 103-111.
30. Davidowa H, Li Y, Plagemann A, (2003) Altered responses to orexigenic (AGRP, MCH) and anorexigenic (alpha-MSH, CART) neuropeptides of paraventricular hypothalamic neurons in early postnatally overfed rats. Eur J Neurosci 18: 613-621.
31. Davidowa H, Plagemann A, (2000) Different responses of ventromedial hypothalamic neurons to leptin in normal and early postnatally overfed rats. Neurosci Lett 293: 21-24.
32. Davidowa H, Plagemann A, (2000) Decreased inhibition by leptin of hypothalamic arcuate neurons in neonatally overfed young rats. Neuroreport 11: 2795-2798.
33. Davidowa H, Plagemann A, (2001) Inhibition by insulin of hypothalamic VMN neurons in rats overweight due to postnatal overfeeding. Neuroreport 12: 3201-3204.
34. Davidowa H, Plagemann A, (2004) Hypothalamic neurons of postnatally overfed, overweight rats respond differentially to corticotropin-releasing hormones. Neurosci Lett 371: 64-68.
35. Levin BE, Govek E, (1998) Gestational obesity accentuates obesity in obesity-prone progeny. Am J Physiol 275: R1374-1379.
36. Ozanne SE, Hales CN, (1999) The long-term consequences of intra-uterine protein malnutrition for glucose metabolism. Proc Nutr Soc 58: 615-619.
37. Srinivasan M, Aalinkeel R, Song F, Mitrani P, Pandya JD, et al. (2006) Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype. Am J Physiol Endocrinol Metab 290: E129-E134.
38. Srinivasan M, Katewa SD, Palaniyappan A, Pandya JD, Patel MS, (2006) Maternal high-fat diet consumption results in fetal malprogramming predisposing to the onset of metabolic syndrome-like phenotype in adulthood. Am J Physiol Endocrinol Metab 291: E792-799.
39. Thone-Reineke C, Kalk P, Dorn M, Klaus S, Simon K, et al. (2006) High-protein nutrition during pregnancy and lactation programs blood pressure, food efficiency, and body weight of the offspring in a sex-dependent manner. Am J Physiol Regul Integr Comp Physiol 291: R1025-1030.
40. Xiao XQ, Williams SM, Grayson BE, Glavas MM, Cowley MA, et al. (2007) Excess weight gain during the early postnatal period is associated with permanent reprogramming of brown adipose tissue adaptive thermogenesis. Endocrinology 148: 4150-4159.
41. Bouret SG, Draper SJ, Simerly RB, (2004) Formation of projection pathways from the arcuate nucleus of the hypothalamus to hypothalamic regions implicated in the neural control of feeding behavior in mice. J Neurosci 24: 2797-2805.
42. Koutcherov Y, Mai JK, Ashwell KW, Paxinos G, (2002) Organization of human hypothalamus in fetal development. J Comp Neurol 446: 301-324.
43. Grayson BE, Allen SE, Billes SK, Williams SM, Smith MS, et al. (2006) Prenatal development of hypothalamic neuropeptide systems in the nonhuman primate. Neuroscience 143: 975-986.
44. Grove KL, Allen S, Grayson BE, Smith MS, (2003) Postnatal development of the hypothalamic neuropeptide Y system. Neuroscience 116: 393-406.
45. Grove KL, Smith MS, (2003) Ontogeny of the hypothalamic neuropeptide Y system. Physiol Behav 79: 47-63.
46. Maltepe E, Bakardjiev AI, Fisher SJ, The placenta: transcriptional, epigenetic, and physiological integration during development. J Clin Invest 120: 1016-1025.
47. McCurdy CE, Bishop JM, Williams SM, Grayson BE, Smith SM, et al. (2009) Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates. The Journal of Clinical Investigation 119: 323-335.
48. Peters JC, (2003) Dietary fat and body weight control. Lipids 38: 123-127.
49. Grayson BE, LeVasseur P, Williams SM, Smith SM, Marks DL, et al. (2007) Changes in Melanocortin Expression and Inflammatory Pathways in Fetal Offspring of Nonhuman Primates Fed a High Fat Diet. in preparation.
50. Sullivan EL, Grayson B, Takahashi D, Robertson N, Maier A, et al. Chronic consumption of a high-fat diet during pregnancy causes perturbations in the serotonergic system and increased anxiety-like behavior in nonhuman primate offspring. J Neurosci 30: 3826-3830.
51. Aagaard-Tillery KM, Grove K, Bishop J, Ke X, Fu Q, et al. (2008) Developmental origins of disease and determinants of chromatin structure: maternal diet modifies the primate fetal epigenome. J Mol Endocrinol 41: 91-102.
52. Suter M, Bocock P, Showalter L, Hu M, Shope C, et al. Epigenomics: maternal high-fat diet exposure in utero disrupts peripheral circadian gene expression in nonhuman primates. Faseb J.
53. Cox J, Williams S, Grove K, Lane RH, Aagaard-Tillery KM, (2009) A maternal high-fat diet is accompanied by alterations in the fetal primate metabolome. Am J Obstet Gynecol 201: 281 e281-289.
54. Lagerstedt SA, Hinrichs DR, Batt SM, Magera MJ, Rinaldo P, et al. (2001) Quantitative determination of plasma c8-c26 total fatty acids for the biochemical diagnosis of nutritional and metabolic disorders. Mol Genet Metab 73: 38-45.
55. Pfaffl MW, (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: e45.
56. ABI (2006) Amplification Efficiency of Taqman Expression Assays.
57. Kidd M, Nadler B, Mane S, Eick G, Malfertheiner M, et al. (2007) GeneChip, geNorm, and gastrointestinal tumors: novel reference genes for real-time PCR. Physiol Genomics 30: 363-370.
58. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, et al. (2002) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 3: RESEARCH0034.
59. Yacoubian S, Serhan CN, (2007) New endogenous anti-inflammatory and proresolving lipid mediators: implications for rheumatic diseases. Nat Clin Pract Rheumatol 3: 570-579; quiz 571 p following 589.
60. Gordon S, (2003) Alternative activation of macrophages. Nat Rev Immunol 3: 23-35.
61. Lumeng CN, Bodzin JL, Saltiel AR, (2007) Obesity induces a phenotypic switch in adipose tissue macrophage polarization. J Clin Invest 117: 175-184.
62. Beckmann CRB, (2006) Obstetrics and gynecology. 5th ed Philadelphia, PA Lippincott Williams & Wilkinspp. xvii, 813 p.
63. Cox LA, Nijland MJ, Gilbert JS, Schlabritz-Loutsevitch NE, Hubbard GB, et al. (2006) Effect of 30 per cent maternal nutrient restriction from 0.16 to 0.5 gestation on fetal baboon kidney gene expression. J Physiol 572: 67-85.
64. Li C, Levitz M, Hubbard GB, Jenkins SL, Han V, et al. (2007) The IGF axis in baboon pregnancy: placental and systemic responses to feeding 70% global ad libitum diet. Placenta 28: 1200-1210.
65. Nijland MJ, Schlabritz-Loutsevitch NE, Hubbard GB, Nathanielsz PW, Cox LA, (2007) Non-human primate fetal kidney transcriptome analysis indicates mammalian target of rapamycin (mTOR) is a central nutrient-responsive pathway. J Physiol 579: 643-656.
66. Schlabritz-Loutsevitch N, Ballesteros B, Dudley C, Jenkins S, Hubbard G, et al. (2007) Moderate maternal nutrient restriction, but not glucocorticoid administration, leads to placental morphological changes in the baboon (Papio sp.). Placenta 28: 783-793.
67. Schlabritz-Loutsevitch NE, Dudley CJ, Gomez JJ, Nevill CH, Smith BK, et al. (2007) Metabolic adjustments to moderate maternal nutrient restriction. Br J Nutr 98: 276-284.
68. Siega-Riz AM, Bodnar LM, Savitz DA, (2002) What are pregnant women eating? Nutrient and food group differences by race. Am J Obstet Gynecol 186: 480-486.
69. Block G, Dresser CM, Hartman AM, Carroll MD, (1985) Nutrient sources in the American diet: quantitative data from the NHANES II survey. II. Macronutrients and fats. Am J Epidemiol 122: 27-40.
70. Subar AF, Krebs-Smith SM, Cook A, Kahle LL, (1998) Dietary sources of nutrients among US children, 1989-1991. Pediatrics 102: 913-923.
71. Simopoulos AP, (1999) Essential fatty acids in health and chronic disease. Am J Clin Nutr 70: 560S-569S.
72. Simopoulos AP, (1991) Omega-3 fatty acids in health and disease and in growth and development. Am J Clin Nutr 54: 438-463.
73. Innis SM, (1991) Essential fatty acids in growth and development. Prog Lipid Res 30: 39-103.
74. Clandinin MT, Chappell JE, Leong S, Heim T, Swyer PR, et al. (1980) Extrauterine fatty acid accretion in infant brain: implications for fatty acid requirements. Early Hum Dev 4: 131-138.
75. Clandinin MT, Chappell JE, Leong S, Heim T, Swyer PR, et al. (1980) Intrauterine fatty acid accretion rates in human brain: implications for fatty acid requirements. Early Hum Dev 4: 121-129.
76. Martinez M, (1992) Tissue levels of polyunsaturated fatty acids during early human development. J Pediatr 120: S129-138.
77. Crabtree JT, Gordon MJ, Campbell FM, Dutta-Roy AK, (1998) Differential distribution and metabolism of arachidonic acid and docosahexaenoic acid by human placental choriocarcinoma (BeWo) cells. Mol Cell Biochem 185: 191-198.
78. Diamant YZ, Metzger BE, Freinkel N, Shafrir E, (1982) Placental lipid and glycogen content in human and experimental diabetes mellitus. Am J Obstet Gynecol 144: 5-11.
79. Hudson DG, Elphick MC, Hull D, (1977) Distribution of label in maternal plasma, placenta, fetal plasma and tissues after injection of 14C-palmitate into the circulation of 21- and 28-day-pregnant rabbits. Biol Neonate 32: 290-295.
80. Ruyle M, Connor WE, Anderson GJ, Lowensohn RI, (1990) Placental transfer of essential fatty acids in humans: venous-arterial difference for docosahexaenoic acid in fetal umbilical erythrocytes. Proc Natl Acad Sci U S A 87: 7902-7906.
81. Herrera E, Amusquivar E, Lopez-Soldado I, Ortega H, (2006) Maternal lipid metabolism and placental lipid transfer. Horm Res 65 (Suppl 3): 59-64.
82. Haggarty P, (2002) Placental regulation of fatty acid delivery and its effect on fetal growth-a review. Placenta 23 (Suppl A): S28-38.
83. Herrera E, Amusquivar E, (2000) Lipid metabolism in the fetus and the newborn. Diabetes Metab Res Rev 16: 202-210.
84. Su HM, Bernardo L, Mirmiran M, Ma XH, Corso TN, et al. (1999) Bioequivalence of dietary alpha-linolenic and docosahexaenoic acids as sources of docosahexaenoate accretion in brain and associated organs of neonatal baboons. Pediatr Res 45: 87-93.
85. Su HM, Corso TN, Nathanielsz PW, Brenna JT, (1999) Linoleic acid kinetics and conversion to arachidonic acid in the pregnant and fetal baboon. J Lipid Res 40: 1304-1312.
86. Su HM, Huang MC, Saad NM, Nathanielsz PW, Brenna JT, (2001) Fetal baboons convert 18:3n-3 to 22:6n-3 in vivo. A stable isotope tracer study. J Lipid Res 42: 581-586.
87. Carlson SE, Cooke RJ, Rhodes PG, Peeples JM, Werkman SH, (1992) Effect of vegetable and marine oils in preterm infant formulas on blood arachidonic and docosahexaenoic acids. J Pediatr 120: S159-167.
88. Olsen SF, Secher NJ, Tabor A, Weber T, Walker JJ, et al. (2000) Randomised clinical trials of fish oil supplementation in high risk pregnancies. Fish Oil Trials In Pregnancy (FOTIP) Team. Bjog 107: 382-395.
89. Neuringer M, Connor WE, Lin DS, Barstad L, Luck S, (1986) Biochemical and functional effects of prenatal and postnatal omega 3 fatty acid deficiency on retina and brain in rhesus monkeys. Proc Natl Acad Sci U S A 83: 4021-4025.
90. Neuringer M, Connor WE, Van Petten C, Barstad L, (1984) Dietary omega-3 fatty acid deficiency and visual loss in infant rhesus monkeys. J Clin Invest 73: 272-276.
91. Aid S, Vancassel S, Poumes-Ballihaut C, Chalon S, Guesnet P, et al. (2003) Effect of a diet-induced n-3 PUFA depletion on cholinergic parameters in the rat hippocampus. J Lipid Res 44: 1545-1551.
92. Kodas E, Vancassel S, Lejeune B, Guilloteau D, Chalon S, (2002) Reversibility of n-3 fatty acid deficiency-induced changes in dopaminergic neurotransmission in rats: critical role of developmental stage. J Lipid Res 43: 1209-1219.
93. Brenner RR, (1981) Nutritional and hormonal factors influencing desaturation of essential fatty acids. Prog Lipid Res 20: 41-47.
94. Castuma JC, Catala A, Brenner RR, (1972) Oxidative desaturation of eicosa-8,11-dienoic acid to eicosa-5,8,11-trienoic acid: comparison of different diets on oxidative desaturation at the 5,6 and 6,7 positions. J Lipid Res 13: 783-789.
95. Eck MG, Wynn JO, Carter WJ, Faas FH, (1979) Fatty acid desaturation in experimental diabetes mellitus. Diabetes 28: 479-485.
96. Inkpen CA, Harris RA, Quackenbush FW, (1969) Differential responses to fasting and subsequent feeding by microsomal systems of rat liver: 6- and 9-desaturation of fatty acids. J Lipid Res 10: 277-282.
97. Rosenthal MD, (1987) Apparent specificity of the thrombin-stimulated deacylation of endothelial glycerolipids for polyunsaturated fatty acids with a delta-5 desaturation. Biochim Biophys Acta 917: 279-289.
98. Sprecher H, (1981) Biochemistry of essential fatty acids. Prog Lipid Res 20: 13-22.
99. Hatano E, (2007) Tumor necrosis factor signaling in hepatocyte apoptosis. J Gastroenterol Hepatol 22 (Suppl 1): S43-44.
100. Ishii H, Adachi M, Fernandez-Checa JC, Cederbaum AI, Deaciuc IV, et al. (2003) Role of apoptosis in alcoholic liver injury. Alcohol Clin Exp Res 27: 1207-1212.
101. Rudiger HA, Clavien PA, (2002) Tumor necrosis factor alpha, but not Fas, mediates hepatocellular apoptosis in the murine ischemic liver. Gastroenterology 122: 202-210.
102. Feldstein AE, Canbay A, Angulo P, Taniai M, Burgart LJ, et al. (2003) Hepatocyte apoptosis and fas expression are prominent features of human nonalcoholic steatohepatitis. Gastroenterology 125: 437-443.
103. Malhi H, Bronk SF, Werneburg NW, Gores GJ, (2006) Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis. J Biol Chem 281: 12093-12101.
104. Malhi H, Barreyro FJ, Isomoto H, Bronk SF, Gores GJ, (2007) Free fatty acids sensitise hepatocytes to TRAIL mediated cytotoxicity. Gut 56: 1124-1131.