Based on the existence of several common pathways and mechanisms of action for renal injury induced by TRI (or its metabolite DCVC) and Hg2+, we hypothesized that prior exposure to one chemical (or its metabolite) would alter the cytotoxic response of kidney cells to a subsequent exposure to the other chemical (or its metabolite). Some of the common pathways include those for metabolism and transport, and the interaction may include processes such as direct competition or induction of an enzyme involved in metabolism. The concept of prior exposure of one chemical producing either an adaptive change in cells or otherwise altering the response of the cell to subsequent exposures to potentially toxic chemicals, is not new. Novel aspects of the present study, however, include our use of primary cultures of hPT cells as a cellular model and comparisons between rodent and human PT cells.
Calabrese and Mehendale (1996) reviewed the role of altered tissue repair as an adaptive strategy to chemical exposures. Of more direct pertinence to the present study, Mehendale and colleagues (Korrapati et al., 2005
; Vaidya et al., 2003a
) demonstrated in a series of studies in mice that enhanced renal cell division is associated with protection of mice from DCVC-induced acute renal failure and death. Their studies have described what they have termed “auto-protection.” In those studies, mice were protected from acute renal injury due to DCVC as a consequence of prior exposures to DCVC. In our study, we have examined the influence of a prior exposure to one chemical on the subsequent toxicity from exposure to another chemical. Any protection that occurs would be described as “hetero-protection.” Besides protection, cells may also experience the opposite response, namely that prior exposure to one chemical may enhance sensitivity to a subsequent exposure to another chemical.
Our initial studies were conducted in rats and rPT cells to further establish effects of Hg2+
on cellular GSH status. In vivo
treatment of rats with a subtoxic dose of Hg2+
(Zalups and Lash, 1990
) produced a number of significant changes in GSH-dependent metabolism in the renal cortex. These changes included increased protein expression of GSTα1 but decreased GSTα2 increased activities of GSH synthesis and GSH-dependent enzymes, and, most significantly for the current study, a significant increase in rates of GSH conjugation of TRI. Analysis of the kinetics of DCVG formation after Hg2+
pretreatment showed that Vmax
was increased but the Km
for TRI was unchanged, suggesting that Hg2+
pretreatment led to more GST being produced. When primary cultures of rPT cells were treated with Hg2+
, a significant increase was observed in GST activity as well.
Assessment of acute cytotoxicity of Hg2+
in rPT cells showed that the dose-response relationship for Hg2+
-induced cell death was very steep, consistent with earlier studies (Lash and Zalups, 1992
; Lash et al., 1999a
). Using LDH inactivation as a measure of acute cellular necrosis, the IC50
was approximately 0.8 μM Hg2+
(cf. ). Analysis of cell cycle distribution and apoptosis by propidium iodide staining and flow cytometry showed that whereas 0.5 μM Hg2+
produced no effect on cell cycle distribution and no significant increase in the fraction of apoptotic cells (cf. ) as compared with control rPT cells, 1 μM Hg2+
caused both a large increase in apoptosis and extensive detachment of cells from the growth surface (cf. ).
Primary cultures of hPT cells, grown under the same conditions and with the same medium as primary cultures of rPT cells, were markedly less responsive to exposures to low concentrations of Hg2+ with respect to cellular GSH status. None of the three isoforms of GST in hPT cells exhibited altered expression after 24-h incubations with Hg2+ (cf. ). Of the GSH-dependent enzyme activities measured, only GRD exhibited a modest increase after a 48-h incubation with Hg2+ (cf. ). In spite of these minimal to modest changes, prior exposure of hPT cells to either Hg2+ or TRI/DCVC had profound effects on necrosis or apoptosis due to subsequent exposures to one of the other chemicals, suggesting that factors in addition to GSH status are important in the response of hPT cells to prior exposures to cytotoxic chemicals.
Pretreatment of hPT cells with between 0.25 μM and 1 μM Hg2+ for 24 h caused a significant increase in necrosis (as determined by LDH inactivation) due to 50 or 200 μM DCVC, but had little effect on that due to TRI (cf. ). In contrast to this response, pretreatment of hPT cells with the same concentrations of Hg2+ markedly decreased apoptosis due to either DCVC or TRI (cf. Figures and ). If one views these two forms of cell death (necrosis or oncosis and apoptosis) as a continuum in the cytotoxic response of cells to toxic chemicals or pathological conditions, then we interpret the results with Hg2+ pretreatment as indicating that sensitivity of hPT cells to TRI or DCVC is enhanced as the mechanism of cell death is shifted from apoptosis to necrosis. In this scenario, cells undergoing apoptosis are viewed as being less seriously compromised than cells undergoing necrosis. The energy dependence and more highly regulated nature of apoptosis would be consistent with this view. Thus, rather than protecting hPT cells from a subsequent exposure to another chemical, prior exposure to low concentrations of Hg2+ enhances the severity of the cellular response to TRI or DCVC.
Unlike the findings with prior exposure to Hg2+, incubation first with either TRI or DCVC generally protected hPT cells from cell death due to Hg2+ (cf. Figures and ). Prior exposure to either TRI or its nephrotoxic metabolite DCVC markedly decreased Hg2+-induced LDH inactivation. Effects on Hg2+-induced apoptosis were, however, somewhat more complex. Whereas prior exposure to TRI clearly increased apoptosis due to Hg2+ at later incubation times, prior exposure to DCVC initially increased the occurrence of Hg2+-induced apoptosis, but then led to a marked decrease at later incubation times. Again, using the view described above of apoptosis and necrosis/oncosis representing a continuum in the cytotoxic response of cells, these results are consistent with prior exposures of hPT primary cell cultures to TRI or DCVC leading to protection from subsequent exposures to Hg2+.
The increased toxicity of TRI in hPT cells after pretreatment with a subtoxic concentration of Hg2+
can readily be rationalized by the ability of Hg2+
to increase bioactivation of TRI by GST and to increase renal PT cell GSH content. Understanding of the mechanism for the increased toxicity of DCVC in hPT cells due to Hg2+
pretreatment requires further study. One possibility is that prior exposure to Hg2+
may induce one or more forms of renal cysteine conjugate β-lyase, as has been shown for the mercapturate of hexachlorobutadiene (Cooper and Pinto, 2006
; MacFarlane et al., 1993
). Similarly, understanding of the mechanism by which pretreatment of hPT cells with TRI or DCVC decreases toxicity of Hg2+
also requires further study, but may involve activation of certain transcription factors and/or some of the protective mechanisms, such as activation of cellular repair and proliferation, as described by Mehendale and colleagues (Korrapati et al., 2005
; Vaidya et al., 2003a
). Some of our earlier results in primary cultures of hPT cells, studying the effects of DCVC on expression of regulatory proteins such as heat shock protein 27 (Hsp27), p53, and nuclear factor κB (NFκB) (Lash et al., 2005
), are consistent with such a potential mechanism. Expression of each of these proteins was induced severalfold by exposure to low concentrations of DCVC. Increased expression of these three proteins is associated with alterations in cellular responses to toxicants, including activation of repair mechanisms and changes in cell cycle status.
In summary, the present study has demonstrated that exposures of hPT cells to one chemical can significantly modulate the sensitivity of the cells to subsequent chemical exposures. The modulation or adaptation of the cells to the initial chemical exposure will depend on the precise mechanisms by which the subsequent chemical produces toxicity. Although changes in cellular GSH status are important, other factors may be more important to the underlying mechanisms by which pretreatment with Hg2+ influences TRI- or DCVC-induced toxicity in the human kidney.