Gap junction-mediated intercellular communication (GJIC) is critical for maintaining integral cellular processes including differentiation and growth control. The disruption of GJIC has been correlated with aberrant function in many cell types, including hepatocytes in vivo; therefore it is imperative that cellular model systems support intercellular communication to simulate normal cellular functions. Functional GJIC has been shown in long-term primary rat hepatocyte cultures, which have been implemented widely to study various aspects of hepatocellular function; however, the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte system. In this report, we analyze GJIC in a dimethylsulfoxide (DMSO)-containing long-term primary mouse hepatocyte culture system. The cells retain morphologic and biochemical characteristics of differentiated hepatocytes through day 30 post plating, including liver-specific gene expression. We further show that connexin32 and connexin26 expression and gap junction plaque formation increase over time in culture concomitant with an increase in GJIC between adjoining primary mouse hepatocytes. In conclusion, the findings described in this study make it possible to maintain differentiated primary mouse hepatocytes that also show GJIC in long-term culture for 30 days. In addition, this system has the potential to be extended to study primary mouse hepatocytes isolated from genetically engineered mice. T he development of in vitro systems to study hepatocellular function largely has been impaired by the inability to maintain hepatocytes in a differentiated state in long-term culture. Previous reports have shown that primary rat hepatocytes, plated on collagencoated dishes and cultured in a chemically defined, serum-free medium supplemented with dimethylsulfoxide (DMSO), retain morphologic and biochemical characteristics of differentiated hepatocytes in long-term culture. 1 DMSO maintains primary rat hepatocytes in a differentiated state over long periods of time (Ͼ60 days), negating the need to coculture the hepatocytes with another cell type. 1 Although the rat system has been invaluable for studying various facets of liver biology in vitro, 1-7 the onset of transgenic technology in murine species has necessitated the development of a primary mouse hepatocyte culture system. Thus, the establishment of a long-term culture system for studying primary mouse hepatocytes (PMHs) would be highly important because hepatocytes could be isolated from genetically engineered mice as well as wild-type mice. Many laboratories have attempted to establish PMH culture systems. Short-term cultures using PMHs have been developed, but these cultures typically are maintained for 24 to 72 hours and therefore either are recovering from cellular isolation procedures or in the process of dedifferentiation. Importantly, gap junctionmediated intercellular communication (GJIC) rapidly declines within the first few days of hepatocyte culture. 8 Previous attempt...
Acetaminophen induced hepatotoxicity has been extensively studied and yet many mechanistic details are still unknown. Our laboratory has established longterm culture conditions for mouse hepatocytes such that the cells establish gap junction mediated intercellular communication and express cytochrome P450s. APAP caused dose dependent and time dependent cytotoxicity in these cells. Prior to inducing toxicity, APAP induced reactive oxygen species (ROS) evaluated by using the dye 2′, 7′‐dichlorofluorescein and MPT measured by two different assays (TMRM/Calcein AM and JC‐1). These hepatocytes can also be iron‐loaded with 3,5,5‐trimethyl hexanoyl ferrocene (TMHF) and depleted of iron by exposure to the iron chelator deferoxamin (DFO). To address the effect of iron on APAP hepatotoxicity, the cells were treated with TMHF for seven days prior to exposure to APAP. Iron loading accelerated the generation of APAP‐induced MPT and cytotoxicity. TMHF treatment alone did not increase ROS and had no effect on MPT. Similarly, when hepatocytes were treated for 48 hours with DFO, reduction in intracellular iron slowed the effects of APAP on MPT and cytotoxicity. We conclude that intracellular hepatic iron levels play a role in APAP‐induced toxicity at the level of MPT. These data suggest that patients with elevated hepatic iron, which occurs in many liver diseases, may be more susceptible to acetaminophen injury (info:ddbj-emblgenbank/DK054482).
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