Alterations in Sensitivity to Estrogen, Dihydrotestosterone, and Xenogens in B-Lymphocytes from Children with Autism Spectrum Disorder and Their Unaffected Twins/Siblings
It has been postulated that androgen overexposure in a susceptible person leads to excessive brain masculinization and the autism spectrum disorder (ASD) phenotype. In this study, the responses to estradiol (E2), dihydrotestosterone (DHT), and dichlorodiphenyldichloroethylene (DDE) on B-lymphocytes from ASD subjects and controls are compared. B cells were obtained from 11 ASD subjects, their unaffected fraternal twins, and nontwin siblings. Controls were obtained from a different cell bank. Lactate dehydrogenase (LDH) and sodium 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction levels were measured after incubation with different concentrations of E2, DHT, and DDE. XTT/LDH ratio, representative of mitochondria number per cell, was calculated. E2, DHT, and DDE all cause “U”-shaped growth curves, as measured by LDH levels. ASD B cells show less growth depression compared to siblings and controls ( ). They also have reduced XTT/LDH ratios ( ) when compared to external controls, whereas siblings had values of XTT/LDH between ASD and external controls. B-lymphocytes from people with ASD exhibit a differential response to E2, DHT, and hormone disruptors in regard to cell growth and mitochondrial upregulation when compared to non-ASD siblings and external controls. Specifically, ASD B-lymphocytes show significantly less growth depression and less mitochondrial upregulation when exposed to these effectors. A mitochondrial deficit in ASD individuals is implied. 1. Introduction Autism spectrum disorder (ASD) is a developmental disorder characterized by abnormal communication, social impairment, and stereotyped restricted interests and behavior. There has been a 10-fold increase in the incidence of ASD over the last two decades, with ASD now affecting 1 in 88 US children [1, 2]. ASD has a 5？:？1 male to female gender bias and is usually diagnosed before 4 years of age. Other male biased conditions [3] including dyslexia [4], specific language impairment, ADHD, and oppositional defiant disorder (ODD) have also increased during the last few decades [3, 4]. The cost of supporting people with ASD in Great Britain, including the cost of lost productivity, is 2.7b/year for children and 25b/year for adults [5]. Medical expenditures on individuals with ASD in the US are 4.1–6.2 greater than for those without, with average annual healthcare expenditures of approximately $6000/child reported and precipitously rising [6, 7]. Phenotypic analyses of male biased cognitive disorders suggest that genetic influences increase susceptibility to
CDC, “Prevalence of autism spectrum disorders—autism and developmental disabilities monitoring network, 14 sites, United States, 2002,” MMWR. Surveillance Summaries: Morbidity and Mortality Weekly Report. Surveillance Summaries, vol. 56, pp. 12–28, 2007.
[3]
M. Rutter, A. Caspi, and T. E. Moffitt, “Using sex differences in psychopathology to study causal mechanisms: unifying issues and research strategies,” Journal of Child Psychology and Psychiatry and Allied Disciplines, vol. 44, no. 8, pp. 1092–1115, 2003.
[4]
J. L. Hawke, R. K. Olson, E. G. Willcut, S. J. Wadsworth, and J. C. DeFries, “Gender ratios for reading difficulties,” Dyslexia, vol. 15, no. 3, pp. 239–242, 2009.
[5]
M. Knapp, R. Romeo, and J. Beecham, “Economic cost of autism in the UK,” Autism, vol. 13, no. 3, pp. 317–336, 2009.
[6]
T. T. Shimabukuro, S. D. Grosse, and C. Rice, “Medical expenditures for children with an autism spectrum disorder in a privately insured population,” Journal of Autism and Developmental Disorders, vol. 38, no. 3, pp. 546–552, 2008.
[7]
D. L. Leslie and A. Martin, “Health care expenditures associated with autism spectrum disorders,” Archives of Pediatrics and Adolescent Medicine, vol. 161, no. 4, pp. 350–355, 2007.
[8]
E. G. Willcutt, R. S. Betjemann, L. M. McGrath et al., “Etiology and neuropsychology of comorbidity between RD and ADHD: the case for multiple-deficit models,” Cortex, vol. 46, no. 10, pp. 1345–1361, 2010.
[9]
C. A. Van Hulle, N. L. Schmidt, and H. H. Goldsmith, “Is sensory over-responsivity distinguishable from childhood behavior problems? A phenotypic and genetic analysis,” Journal of Child Psychology and Psychiatry and Allied Disciplines, vol. 53, no. 1, pp. 64–72, 2012.
[10]
P. L. Morgan, G. Farkas, P. A. Tufis, and R. A. Sperling, “Are reading and behavior problems risk factors for each other?” Journal of Learning Disabilities, vol. 41, no. 5, pp. 417–436, 2008.
[11]
J. Van Honk, D. J. Schutter, P. A. Bos, A.-W. Kruijt, E. G. Lentjes, and S. Baron-Cohen, “Testosterone administration impairs cognitive empathy in women depending on second-to-fourth digit ratio,” Proceedings of the National Academy of Sciences of the United States of America, vol. 108, no. 8, pp. 3448–3452, 2011.
[12]
J. T. Manning, D. Scutt, J. Wilson, and D. I. Lewis-Jones, “The ratio of 2nd to 4th digit length: a predictor of sperm numbers and concentrations of testosterone, luteinizing hormone and oestrogen,” Human Reproduction, vol. 13, no. 11, pp. 3000–3004, 1998.
[13]
I. Gigli and L. E. Bussmann, “Exercise and ovarian steroid hormones: their effects on mitochondrial respiration,” Life Sciences, vol. 68, no. 13, pp. 1505–1514, 2001.
[14]
J. D. Y. Chow, M. E. E. Jones, K. Prelle, E. R. Simpson, and W. C. Boon, “A selective estrogen receptor α agonist ameliorates hepatic steatosis in the male aromatase knockout mouse,” Journal of Endocrinology, vol. 210, no. 3, pp. 323–334, 2011.
[15]
C. M. Klinge, “Estrogenic control of mitochondrial function and biogenesis,” Journal of Cellular Biochemistry, vol. 105, no. 6, pp. 1342–1351, 2008.
[16]
R. W. Irwin, J. Yao, R. T. Hamilton, E. Cadenas, R. D. Brinton, and J. Nilsen, “Progesterone and estrogen regulate oxidative metabolism in brain mitochondria,” Endocrinology, vol. 149, no. 6, pp. 3167–3175, 2008.
[17]
A. Saborido, J. Vila, F. Molano, and A. Megias, “Effect of anabolic steroids on mitochondria and sarcotubular system of skeletal muscle,” Journal of Applied Physiology, vol. 70, no. 3, pp. 1038–1043, 1991.
[18]
R. Seifeddine, A. Dreiem, C. Tomkiewicz et al., “Hypoxia and estrogen co-operate to regulate gene expression in T-47D human breast cancer cells,” Journal of Steroid Biochemistry and Molecular Biology, vol. 104, no. 3-5, pp. 169–179, 2007.
[19]
L. Rato, M. G. Alves, S. Socorro, R. A. Carvalho, J. E. Cavaco, and P. F. Oliveira, “Metabolic modulation induced by oestradiol and DHT in immature rat Sertoli cells cultured in vitro,” Bioscience Reports, vol. 32, no. 1, pp. 61–69, 2012.
[20]
D. J. Sheskin, Handbook of Parametric and Nonparametric Statistical Procedures, Chapman & Hall/CRC, London, UK, 4th edition, 2007.
[21]
R. Anney, L. Klei, D. Pinto et al., “A genome-wide scan for common alleles affecting risk for autism,” Human Molecular Genetics, vol. 19, no. 20, pp. 4072–4082, 2010.
[22]
D. Pinto, A. T. Pagnamenta, L. Klei et al., “Functional impact of global rare copy number variation in autism spectrum disorders,” Nature, vol. 466, no. 7304, pp. 368–372, 2010.
[23]
V. W. Hu, T. Sarachana, S. K. Kyung et al., “Gene expression profiling differentiates autism case-controls and phenotypic variants of autism spectrum disorders: evidence for circadian rhythm dysfunction in severe autism,” Autism Research, vol. 2, no. 2, pp. 78–97, 2009.
[24]
V. W. Hu, A. T. Nguyen, K. S. Kim et al., “Gene expression profiling of lymphoblasts from autistic and nonaffected sib pairs: altered pathways in neuronal development and steroid biosynthesis,” PLoS ONE, vol. 4, no. 6, Article ID e5775, 2009.
[25]
M. Pierdominici, A. Maselli, T. Colasanti et al., “Estrogen receptor profiles in human peripheral blood lymphocytes,” Immunology Letters, vol. 132, no. 1-2, pp. 79–85, 2010.
[26]
N. Kanda, T. Tsuchida, and K. Tamaki, “Testosterone inhibits immunoglobulin production by human peripheral blood mononuclear cells,” Clinical and Experimental Immunology, vol. 106, no. 2, pp. 410–415, 1996.
[27]
Z. Zhou, C. H. L. Shackleton, S. Pahwa, P. C. White, and P. W. Speiser, “Prominent sex steroid metabolism in human lymphocytes,” Molecular and Cellular Endocrinology, vol. 138, no. 1-2, pp. 61–69, 1998.
[28]
E. Dewailly, P. Ayotte, and S. Dodin, “Could the rising levels of estrogen receptor in breast cancer be due to estrogenic pollutants?” Journal of the National Cancer Institute, vol. 89, no. 12, pp. 888–889, 1997.
[29]
S.-I. Narita, R. M. Goldblum, C. S. Watson et al., “Environmental estrogens induce mast cell degranulation and enhance IgE-mediated release of allergic mediators,” Environmental Health Perspectives, vol. 115, no. 1, pp. 48–52, 2007.
[30]
I. Hertz-Picciotto and L. Delwiche, “The rise in autism and the role of age at diagnosis,” Epidemiology, vol. 20, no. 1, pp. 84–90, 2009.
[31]
S. M. Rhind, “Anthropogenic pollutants: a threat to ecosystem sustainability?” Philosophical Transactions of the Royal Society B, vol. 364, no. 1534, pp. 3391–3401, 2009.
[32]
M. Larsson, B. Weiss, S. Janson, J. Sundell, and C.-G. Bornehag, “Associations between indoor environmental factors and parental-reported autistic spectrum disorders in children 6–8 years of age,” NeuroToxicology, vol. 30, no. 5, pp. 822–831, 2009.
[33]
T. Decker and M.-L. Lohmann-Matthes, “A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity,” Journal of Immunological Methods, vol. 115, no. 1, pp. 61–69, 1988.
[34]
J.-H. Lu, Y.-T. Chiu, H.-W. Sung et al., “XTT-colorimetric assay as a marker of viability in cryoprocessed cardiac valve,” Journal of Molecular and Cellular Cardiology, vol. 29, no. 4, pp. 1189–1194, 1997.
[35]
C. Korzeniewski and D. M. Callewaert, “An enzyme-release assay for natural cytotoxicity,” Journal of Immunological Methods, vol. 64, no. 3, pp. 313–320, 1983.
[36]
T. Decker and M.-L. Lohmann-Matthes, “A quick and simple method for the quantitation of lactate dehydrogenase release in measurements of cellular cytotoxicity and tumor necrosis factor (TNF) activity,” Journal of Immunological Methods, vol. 115, no. 1, pp. 61–69, 1988.
[37]
M. V. Berridge, P. M. Herst, and A. S. Tan, “Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction,” Biotechnology Annual Review, vol. 11, pp. 127–152, 2005.
[38]
O. Huet, J. M. Petit, M. H. Ratinaud, and R. Julien, “NADH-dependent dehydrogenase activity estimation by flow cytometric analysis of 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction,” Cytometry, vol. 13, no. 5, pp. 532–539, 1992.
[39]
J. Gorski and F. Gannon, “Current models of steroid hormone action: a critique,” Annual Review of Physiology, vol. 38, pp. 425–450, 1976.
[40]
J. A. Taylor, C. A. Richter, R. L. Ruhlen, and F. S. Vom Saal, “Estrogenic environmental chemicals and drugs: mechanisms for effects on the developing male urogenital system,” Journal of Steroid Biochemistry and Molecular Biology, vol. 127, no. 1-2, pp. 83–95, 2011.
[41]
J. Garcia-Estrada, J. A. Del Rio, S. Luquin, E. Soriano, and L. M. Garcia-Segura, “Gonadal hormones down-regulate reactive gliosis and astrocyte proliferation after a penetrating brain injury,” Brain Research, vol. 628, no. 1-2, pp. 271–278, 1993.
[42]
C. S. Casley, L. Canevari, J. M. Land, J. B. Clark, and M. A. Sharpe, “β-amyloid inhibits integrated mitochondrial respiration and key enzyme activities,” Journal of Neurochemistry, vol. 80, no. 1, pp. 91–100, 2002.
[43]
C. S. Casley, J. M. Land, M. A. Sharpe, J. B. Clark, M. R. Duchen, and L. Canevari, “β-amyloid fragment 25-35 causes mitochondrial dysfunction in primary cortical neurons,” Neurobiology of Disease, vol. 10, no. 3, pp. 258–267, 2002.
[44]
T.-V. V. Nguyen, M. Yao, and C. J. Pike, “Dihydrotestosterone activates CREB signaling in cultured hippocampal neurons,” Brain Research, vol. 1298, pp. 1–12, 2009.
[45]
S. Baron-Cohen, “The extreme male brain theory of autism,” Trends in Cognitive Sciences, vol. 6, no. 6, pp. 248–254, 2002.
[46]
B. Auyeung, S. Baron-Cohen, E. Ashwin, R. Knickmeyer, K. Taylor, and G. Hackett, “Fetal testosterone and autistic traits,” British Journal of Psychology, vol. 100, no. 1, pp. 1–22, 2009.
[47]
G. G. J. M. Kuiper, B. Carlsson, K. Grandien et al., “Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors and α and β,” Endocrinology, vol. 138, no. 3, pp. 863–870, 1997.
[48]
H. Asperger, “Die “Autistischen Psychopathen” im Kindesalter,” Archiv für Psychiatrie und Nervenkrankheiten, vol. 117, no. 1, pp. 76–136, 1944.
[49]
M. H. McIntyre, “The use of digit ratios as markers for perinatal androgen action,” Reproductive Biology and Endocrinology, vol. 4, article 10, 2006.
[50]
E. I. De Bruin, F. Verheij, T. Wiegman, and R. F. Ferdinand, “Differences in finger length ratio between males with autism, pervasive developmental disorder-not otherwise specified, ADHD, and anxiety disorders,” Developmental Medicine & Child Neurology, vol. 48, no. 12, pp. 962–965, 2006.
[51]
E. I. De Bruin, P. F. A. De Nijs, F. Verheij, D. H. Verhagen, and R. F. Ferdinand, “Autistic features in girls from a psychiatric sample are strongly associated with a low 2D:4D ratio,” Autism, vol. 13, no. 5, pp. 511–521, 2009.
[52]
C. Jackson, “Prediction of hemispheric asymmetry as measured by handedness from digit length and 2D:4D digit ratio,” Laterality, vol. 13, no. 1, pp. 34–50, 2008.
[53]
J. M. Lust, R. H. Geuze, C. Van de Beek, P. T. Cohen-Kettenis, A. Bouma, and T. G. G. Groothuis, “Differential effects of prenatal testosterone on lateralization of handedness and language,” Neuropsychology, vol. 25, no. 5, pp. 581–589, 2011.
[54]
J. A. Hauck and D. Dewey, “Hand preference and motor functioning in children with autism,” Journal of Autism and Developmental Disorders, vol. 31, no. 3, pp. 265–277, 2001.
[55]
K. M. Colby and C. Parkison, “Handedness in autistic children,” Journal of Autism and Childhood Schizophrenia, vol. 7, no. 1, pp. 3–9, 1977.
[56]
E. E. Maccoby and C. N. Jacklin, The Psychology of Sex Differences, Stanford University Press, Stanford, Calif, USA, 1974.
[57]
D. Voyer, S. Voyer, and M. P. Bryden, “Magnitude of sex differences in spatial abilities: a meta-analysis and consideration of critical variables,” Psychological Bulletin, vol. 117, no. 2, pp. 250–270, 1995.
[58]
I. Soulières, T. A. Zeffiro, M. L. Girard, and L. Mottron, “Enhanced mental image mapping in autism,” Neuropsychologia, vol. 49, no. 5, pp. 848–857, 2011.
[59]
L. Martini, “The 5alpha-reduction of testosterone in the neuroendocrine structures. Biochemical and physiological implications,” Endocrine Reviews, vol. 3, no. 1, pp. 1–25, 1982.
[60]
Z. Weihua, R. Lathe, M. Warner, and J.-？. Gustafsson, “An endocrine pathway in the prostate, ERβ, AR, 5α-androstane-3β,17β-diol, and CYP7B1, regulates prostate growth,” Proceedings of the National Academy of Sciences of the United States of America, vol. 99, no. 21, pp. 13589–13594, 2006.
[61]
T. R. Pak, W. C. J. Chung, L. R. Hinds, and R. J. Handa, “Estrogen receptor-β mediates dihydrotestosterone-induced stimulation of the arginine vasopressin promoter in neuronal cells,” Neuroendocrinology, vol. 148, no. 7, pp. 3371–3382, 2007.
[62]
M. Hines, S. F. Ahmed, and I. A. Hughes, “Psychological outcomes and gender-related development in complete androgen insensitivity syndrome,” Archives of Sexual Behavior, vol. 32, no. 2, pp. 93–101, 2003.
[63]
S. A. Berenbaum, K. K. Bryk, N. Nowak, C. A. Quigley, and S. Moffat, “Fingers as a marker of prenatal androgen exposure,” Endocrinology, vol. 150, no. 11, pp. 5119–5124, 2009.
[64]
J. Imperato-McGinley, R. E. Peterson, T. Gautier, and E. Sturla, “Androgens and the evolution of male-gender identity among male pseudohermaphrodites with 5α-reductase deficiency,” The New England Journal of Medicine, vol. 300, no. 22, pp. 1233–1237, 1979.
[65]
M. E. E. Jones, W. C. Boon, J. Proietto, and E. R. Simpson, “Of mice and men: the evolving phenotype of aromatase deficiency,” Trends in Endocrinology & Metabolism, vol. 17, no. 2, pp. 55–64, 2006.
[66]
L. Lin, O. Ercan, J. Raza et al., “Variable phenotypes associated with aromatase (CYP19) insufficiency in humans,” The Journal of Clinical Endocrinology and Metabolism, vol. 92, no. 3, pp. 982–990, 2007.
[67]
D. A. Puts, M. A. McDaniel, C. L. Jordan, and S. M. Breedlove, “Spatial ability and prenatal androgens: meta-analyses of congenital adrenal hyperplasia and digit ratio (2D:4D) studies,” Archives of Sexual Behavior, vol. 37, no. 1, pp. 100–111, 2008.
[68]
A. ？kten, M. Kalyoncu, and N. Yari？, “The ratio of second- and fourth-digit lengths and congenital adrenal hyperplasia due to 21-hydroxylase deficiency,” Early Human Development, vol. 70, no. 1-2, pp. 47–54, 2002.
[69]
R. Nass, S. Baker, and P. Speiser, “Hormones and handedness: left-hand bias in female congenital adrenal hyperplasia patients,” Neurology, vol. 37, no. 4, pp. 711–715, 1987.
[70]
L. Starka, I. Cermakova, M. Duskova, M. Hill, M. Dolezal, and V. Polacek, “Hormonal profile of men with premature balding,” Experimental and Clinical Endocrinology & Diabetes, vol. 112, pp. 24–28, 2004.
[71]
A. M. Hillmer, A. Flaquer, S. Hanneken et al., “Genome-wide scan and fine-mapping linkage study of androgenetic alopecia reveals a locus on chromosome 3q26,” The American Journal of Human Genetics, vol. 82, no. 3, pp. 737–743, 2008.
[72]
K. Allen-Brady, J. Miller, N. Matsunami et al., “A high-density SNP genome-wide linkage scan in a large autism extended pedigree,” Molecular Psychiatry, vol. 14, no. 6, pp. 590–600, 2009.
[73]
D. A. Geier, H. A. Young, and M. R. Geier, “Thimerosal exposure and increasing trends of premature puberty in the vaccine safety datalink,” Indian Journal of Medical Research, vol. 131, no. 4, pp. 500–507, 2010.
[74]
M. A. Sharpe, T. L. Gist, and D. S. Baskin, “B-Lymphocytes from a population of children with autism spectrum disorder and their unaffected siblings exhibit hypersensitivity to thimerosal,” Journal of Toxicology, vol. 2013, Article ID 801517, 11 pages, 2013.
