Alterations in sensitivity to estrogen, dihydrotestosterone, and xenogens in B-lymphocytes from children with autism spectrum disorder and their unaffected twins/siblings

Abstract

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 (P < 0.01). They also have reduced XTT/LDH ratios (P < 0.01) 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.

Figure 1

Changes in growth of B-lymphocytes and in their mitochondrial/cell ratio as a function of E2, DHT, and DDE concentrations. The figure shows the changes observed in B cells grown in the presence of E2, DHT, and DDE. The upper part of each panel shows the populations sorted by gender, with the 10 male AUT (⚫), 10 Con (□), 10 Bro (▲), and 10 Sis (∆). The lower part of each panel shows the populations sorted by birth relationship, with the average of 11 AUT (⚫), 11 Con (□), 11 Twin (▲), and 11 Sib (∆). The solid and dashed lines represent the AUT and Bro/Twin plots, respectively. (a) shows change in growth of B cells (LDH levels), and (b) shows change in the mitochondria/cell ratio (XTT/LDH), after five days in presence of effectors at listed concentrations as compared to controls. The solid line connects the AUT and the broken line the Bro and Twin, respectively. Error bars represent SEM. ASD: autism spectrum disorder; AUT: B cells from individual with ASD; Bro: B cells from brother of individual with ASD; Con: control B cells from individual with no personal or family history of ASD; DDE: dichlorodiphenyldichloroethylene; DHT: dihydrotestosterone; E2: estradiol; LDH: lactate dehydrogenase; SEM: standard error of the mean; Sib: B cells from phenotypically normal male or female siblings of individual with ASD; Sis: B cells from phenotypically normal sister of individual with ASD; Twin: B cells from phenotypically normal fraternal twin of individual with ASD; XTT: 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; XTT/LDH: ratio of XTT/LDH indicative of mitochondrial function per cell.

Figure 2

Changes in growth of B-lymphocytes and their mitochondrial/cell ratio as a function of E2, DHT, and DDE concentrations. Cumulative sum plots of Figure 1 data were created showing the effects of the 3 effectors on the 4 cell types, AUT (⚫), Bro (▲), Sis (∆), and Con (□). Solid line = AUT, broken line = Bro. Error bars are SEM. (a) shows changes in cellular growth as evidenced by LDH levels in the different cell populations when grown in the presence of the 3 effectors. (b) shows changes in the mitochondrial function per cell as evidenced by the XTT/LDH ratio. The starting concentration of zero nM represents the baseline LDH and XTT/LDH levels normalized to 0. The most significant difference between the AUT and Con cells in regard to growth suppression and mitochondrial function was seen in the presence of DHT. ASD: autism spectrum disorder; AUT: B cells from individual with ASD; Bro: B cells from brother of individual with ASD; Con: control B cells from individual with no personal or family history of ASD; DDE: dichlorodiphenyldichloroethylene; DHT: dihydrotestosterone; E2: estradiol; LDH: lactate dehydrogenase; SEM: standard error of the mean; Sis: B cells from phenotypically normal sister of individual with ASD; XTT: 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; XTT/LDH: ratio of XTT/LDH indicative of mitochondrial function per cell.

Figure 3

Effects of E2, DHT, and DDE on normal human astrocyte and human cortical neuron growth and XTT/LDH ratio. The figure shows the changes in normal human astrocyte (black bars) and human cortical neuron (white bars) growth (LDH level, left column) and the XTT/LDH ratio (right column) over four days in the presence of 1.2 nM E2, 12 nM DHT, and 50 nM DDE. Each bar represents 15 individual wells of cells and the error bars are the SD. E2, DHT, and DDE inhibit astrocytic and neuronal cell growth and cause changes in mitochondrial levels. DDE: dichlorodiphenyldichloroethylene; DHT: dihydrotestosterone; E2: estradiol; LDH: lactate dehydrogenase; SD: standard deviation; XTT: 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; XTT/LDH: ratio of XTT/LDH indicative of mitochondrial function per cell.