Arsenite Effects on Mitochondrial Bioenergetics in Human and Mouse Primary Hepatocytes Follow a Nonlinear Dose Response

Abstract

Arsenite is a known carcinogen and its exposure has been implicated in a variety of noncarcinogenic health concerns. Increased oxidative stress is thought to be the primary cause of arsenite toxicity and the toxic effect is thought to be linear with detrimental effects reported at all concentrations of arsenite. But the paradigm of linear dose response in arsenite toxicity is shifting. In the present study we demonstrate that arsenite effects on mitochondrial respiration in primary hepatocytes follow a nonlinear dose response. In vitro exposure of primary hepatocytes to an environmentally relevant, moderate level of arsenite results in increased oxidant production that appears to arise from changes in the expression and activity of respiratory Complex I of the mitochondrial proton circuit. In primary hepatocytes the excess oxidant production appears to elicit adaptive responses that promote resistance to oxidative stress and a propensity to increased proliferation. Taken together, these results suggest a nonlinear dose-response characteristic of arsenite with low-dose arsenite promoting adaptive responses in a process known as mitohormesis, with transient increase in ROS levels acting as transducers of arsenite-induced mitohormesis.

Figure 1

Nonlinear dose response of arsenite in primary hepatocytes. Growth and survival response of (a) mouse primary hepatocytes, (b) human primary hepatocytes, and (c) human hepatoma cell line HepG2 exposed to increasing concentration of sodium arsenite for 16 h. (d) Arsenite IC₅₀ values for HepG2, mouse primary hepatocytes, and human primary hepatocytes. Growth and survival were measured using the MTT assay as described in methods. Results are representative of three independent experiments. The insets in (a), (b), and (c) show the toxicity profile at arsenite concentrations from 0 to 10 μM. Values represent mean ± SD. Survival values were significantly different from vehicle treated control cells; P < 0.01.

Figure 2

Arsenite effect on mitochondrial bioenergetics in primary hepatocytes. Mitochondrial bioenergetic profile of (a) mouse and (b) human primary hepatocytes in response to increasing concentration of arsenite. Primary hepatocytes in the presence of glucose were exposed sequentially to oligomycin (OLIGO), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (ROT/ANTI A) as described in methods. Nonmitochondrial respiration after the final addition of ROT/ANTI A was subtracted from the other values. BR, basal respiration; OLIGO, oligomycin-sensitive or ATP-linked respiration; FCCP, maximal respiration in the presence of FCCP. Tables to the right of (a) and (b) provide the statistical analysis of the bioenergetic profile. Results representative of 5 independent experiments. Each independent experiment was run using an independent mouse and an independent human liver (representing 5 different mouse and 5 different human livers). Bioenergetic profile data presented as mean ± SD.

Figure 3

Arsenite effect on mitochondrial respiratory complex expression and activity in primary hepatocytes. ((a) and (c)) Immunoblot analysis of mitochondrial respiratory complex expression in (a) mouse and (c) human primary hepatocytes. Histograms to the right of (a) and (c) show ImageJ analysis of the respective immunoblots. ((b) and (d)) Complex I activity in (b) mouse and (d) human primary hepatocytes. Tables to the right of (b) and (d) provide statistical analysis of Complex I activity data, respectively. Results representative of four independent experiments. Complex I activity data presented as mean ± SD. ImageJ analysis data are normalized to actin and presented as mean ± SD. ^∗Significantly different from vehicle and 0–2.5 μM arsenite treated hepatocytes; P < 0.01. ^#Significantly different from 5 μM arsenite treated hepatocytes; P < 0.01. C-I, Complex I; C-IV, Complex IV; C-III, Complex III; C-V, Complex V.

Figure 4

Arsenite induced reactive oxygen radicals (ROS) in hepatocytes. ((a) and (c)) Live cell imaging of MitoSOX fluorescence and ((b) and (d)) spectrofluorometric analysis of mitochondrial reactive oxygen production (MitoSOX fluorescence) in lysates of ((a) and (b)) mouse and ((c) and (d)) human primary hepatocytes. Mitochondrial localization of MitoSOX was confirmed using MitoTracker (mitochondria). Overlay (yellow) represents colocalization of MitoSOX and MitoTracker. Results representative of three independent experiments. ^∗Significantly different from vehicle control cells; P < 0.01. ^#Significantly different from 0.3 and 0.6 μM arsenite treated cells; P < 0.05. ^$Significantly different from 2.5 μM arsenite treated cells; P < 0.05. ^&Significantly different from 5 μM arsenite treated cells; P < 0.01. Values in (b) and (d) represent mean ± SD. APAP, acetaminophen.

Figure 5

Adaptive response to arsenite in primary hepatocytes. ((a) and (b)) Immunoblot analysis of heme oxygenase (HO1), catalase, and superoxide dismutase 2 (SOD2) expression in (a) mouse and (b) human primary hepatocytes. Histograms to the right of (a) and (b) show ImageJ analysis of the immunoblot data presented in (a) and (b), respectively. Results representative of three independent experiments. ImageJ analysis data are normalized to actin and presented as mean ± SD. ^∗Significantly different from vehicle treated cells; P < 0.01. ^#Significantly different from 0.3 uM arsenite treated cells; P < 0.01. ^$Significantly different from 0.6 and 1.2 uM arsenite treated cells in panel (a); P < 0.01. ^&Significantly different from 1.2 uM arsenite treated cells in panel (b); P < 0.01. ^@Significantly different from 10 uM arsenite treated cell in panel (b); P < 0.01.

Figure 6

Arsenite induces a primary hepatocyte phenotype that resists oxidant stress. Arsenic and vehicle alone treated (a) mouse and (b) human primary hepatocytes were exposed to a toxic oxidative stress from 100 μM H₂O₂. Cells from both groups were exposed to H₂O₂ at time = 0 and necrosis was determined using SYTOX Green over 72 h following exposure. Data presented as mean ± SD. ^∗Significantly different from vehicle treated hepatocytes at the designated time point; P < 0.01. ^#Significantly different from 10 uM arsenic treated hepatocytes at the designated time point: P < 0.01.

Figure 7

Arsenite effects on mouse hepatocyte proliferation. Mouse hepatocytes exposed to arsenite for 16 h on day 2 of culture were treated with hepatocyte growth factor on day 3. Proliferation in response to arsenite and HGF was evaluated by incorporation of bromodeoxyuridine (BRDU) into DNA. y-axis indicates percent of labeled nuclei due to incorporation of BRDU into DNA. Results presented as mean ± SD. Results representative of two independent experiments. HGF positive stain was significantly different from vehicle and arsenic only treated cells: P < 0.01.