化学物诱发肝脏毒性过程中适应性反应的分子基础
Molecular Basis for Adaptive Responses during ChemicallyInduced Hepatotoxicity
Philip C. Burcham1
Pharmacology Unit, School of Medicine and Pharmacology,
the University of Western Australia, Nedlands, Western
Australia 6009, Australia
Prior exposure to toxic xenobiotics can elicit changes
within liver that confer resistance upon subsequent
reexposure— a phenomenon sometimes referred to as
autoprotection (Dalhoff et al., 2001; Thakore and
Mehendale, 1991). Classically, the mechanisms underlying
such responses have been assigned to either of two
categories. First, so-called toxicodynamic alterations
involve molecular changes within hepatocytes that
counteract the deleterious biochemical events whereby
hepatotoxicants induce toxicity. An example of this
phenomenon is the induction of various antioxidant
pathways in rodent liver on exposure to such diverse
prooxidants as heavy metals, plasticizers, and
hepatotoxic drugs (Bando et al., 2005; Nicholls-Grzemski
et al., 2000; O’Brien, et al., 2000). Second,
dispositional or toxicokinetic alterations decrease the
delivery of chemicals to the liver or otherwise diminish
the intracellular concentrations of xenobiotics attained
in hepatocytes. In the past, the mechanisms underlying
this second class of adaptive changes were often largely
unknown. Together with other recent work from the
Manautou group and other laboratories, the report by
Aleksunes et al. in this issue of Toxicological Sciences
shows how our understanding of the molecular basis for
these complex phenomena is steadily improving.
Studying changes to the abundance of mRNA transcripts
during chemical exposure holds promise for illuminating
the biochemical mechanisms underlying a wide range of
toxicological syndromes (Hayes and Bradfield, 2005). Use
of cDNA or oligo microarrays to characterize thousands
of mRNA transcripts simultaneously is a common approach,
although technologies that focus on gene subsets of
known toxicological relevance present a useful
alternative strategy. In a companion study to the
present work, Aleksunes et al. (2005) used branched DNA
signal amplification to quantify mRNAs for one dozen
membrane transporters in the livers of mice 6, 24, and
48 h after they received moderately hepatotoxic doses of
acetaminophen or carbon tetrachloride (Aleksunes et al.,
2005). These classic hepatoxicants induce liver cell
injury following conversion to their respective reactive
intermediates, N-acetyl-p-quinoneimine and the
trichloromethyl radical. Suggesting an attempt by
hepatocytes to minimize uptake of noxious substances
from the sinusoidal circulation, toxic doses of
acetaminophen decreased mRNA transcripts for various
basolateral uptake transporters including organic
anion-transporting polypeptides (Oatp1a1, Oatp1b2) and
the sodium/ taurocholate-cotransporting peptide (Ntcp)
(Aleksunes et al., 2005). A concurrent effort by
acetaminophen- and CCl4- intoxicated hepatocytes to
increase compound efflux was indicated by the elevated
transcripts for genes involved in canalicular transport
into bile (Mrp2) and also for returning chemicals to the
general circulation (the basolateral efflux transporters
Mrp 1, 3, 4) (Aleksunes et al., 2005).
Although the power of transcript-profiling is readily
apparent, use of this technology to characterize gene
profiles in rodent models of chemical toxicity can be
confounded by such technical challenges as
irreproducibility, unwanted signal variation, and noise
(Fielden and Zacharewski, 2001). In a similar manner,
the results obtained by Aleksunes et al. (2005) were
plagued by an unwanted degree of inconsistency. For
example, while hepatic levels of the Oatp1a1 mRNA
transcript were strongly suppressed (_90%) in mice 48 h
after they received 400 mg/kg acetaminophen, this
transcript was unaltered by this dose at the 24 h time
point, despite the fact that a lower dose of
acetaminophen (300 mg/kg) reduced this marker by _60% at
the earlier time (Aleksunes et al., 2005). Furthermore,
in mice that received the top dose of acetaminophen, the
Mrp2 and Mrp3 genes were strongly induced at 6 and 48 h,
yet were at comparable levels to controls at an
intermediate time point (i.e., 24 h, see Fig. 4 in
Aleksunes et al., 2005). Together with the more
fundamental concern of whether altered mRNA levels for a
given gene translate to corresponding changes in protein
abundance, these frustrating outcomes highlight some of
the challenges accompanying the use of
transcript-profiling during toxicological research.
The latest work by Aleksunes et al. in this issue of
Toxicological Sciences addresses some of these issues by
using immunohistochemistry to assess the levels of
selected membrane transporter proteins in the livers of
acetaminophen and CCl4-treated mice. Consistent with
regulation at the transcriptional level as a primary
determinant of protein abundance, exposure-related
changes to the levels of several transporter proteins
were observed. Moreover, these trends correlated well
with measurements of the corresponding mRNA transcripts
in the preceding work. In particular, a striking
induction of the sinusoidal ATP-dependent transporter
Mrp4 was detected in both acetaminophen and CCl4-treated
mouse livers, with the fold increases over controls
equal to or exceeding those observed for Mrp4 mRNA in
the earlier study. Mrp4 protein was barely detectable in
control livers, yet increased strongly in centrilobular
hepatocytes 24 and 48 h after mice received
acetaminophen and CCl4. At the highest dose of CCl4
(i.e. 25 ll/kg), the degree of induction was,
respectively, 7- and 26-fold at the 24 and 48 h time
points. Mrp4 induction was typically localized to rings
of perivenous hepatocytes, although the diffuse nature
of the immunostaining suggested disruption of
intracellular Mrp4 trafficking at the top dose of CCl4.
These intriguing observations seem likely to cultivate
further fruitful research activity. An obvious issue is
the functional significance of the present findings,
since while alterations to membrane transporters could
conceivably diminish toxicity upon reexposure to
hepatotoxicants, the degree to which these changes
confer autoprotection needs clarification. A range of
mechanisms are thought to contribute to both
acetaminophenand CCl4-induced autoprotection, including
elevated tissue repair, mitogenesis, and hepatocellular
regeneration (Grunnet et al., 2003; Thakore and
Mehendale, 1991). The relative importance of altered
transporter expression relative to these other
autoprotective mechanisms is an unanswered question that
will require careful attention to experimental design
during future work.
Second, the strong upregulation of Mrp4 during
chemically induced hepatic injury raises the question as
to which intracellular substrates are high priorities
for removal by this pathway in injured hepatocytes. The
list of xenobiotics that are known Mrp4 substrates is
quite short but may include various nucleotide or base
analogues used in the treatment of cancer or viral
infections, although the physiological relevance of Mrp4
to the clearance of these drugs has been questioned
(Reid et al., 2003). Given its limited role in
sinusoidal xenobiotic export, the overexpression of Mrp4
seems unlikely to represent an effort by the liver to
clear the hepatotoxicants used in these experiments
(acetaminophen and CCl4). In contrast, a number of
endogenous substances do appear to be transported by
Mrp4, including various eicosanoids derived from
arachidonic acid (e. g., prostaglandins E1 and E2) (Reid
et al., 2003). The Mrp4 pathway may also serve as a
low-affinity, ‘‘overflow’’ transporter for cyclic
nucleotides under conditions where phosphodiesterase
activity is limiting (Wielinga et al., 2003).
The recent finding that Mrp4 efficiently transports
sulfated bile acids may be particularly relevant to its
induction during chemically induced hepatotoxicity.
Under cholestatic conditions where apical bile acid
export is impaired, compensatory Mrp4 up-regulation
accompanied by sulfotransferase induction may offset
hepatic accumulation of these toxic species, thereby
explaining the strong increases in serum sulfated bile
acids during experimental cholestasis in rats (Zelcer et
al., 2003). Abnormalities in serum bile acids have long
been known to accompany poisoning syndromes involving
acetaminophen and CCl4 (Anwer et al., 1976; James et
al., 1975). Future studies could seek to clarify the
contribution of hepatic Mrp4 induction to the altered
plasma profiles of monoanionic and dianionic bile acids
seen in hepatotoxicant-treated animals.
Another question raised by the new work concerns the
mechanism whereby reactive intermediates as diverse as
the quinoneimine derived from acetaminophen and the free
radical formed from CCl4 can trigger the common
induction of a membrane transporter such as Mrp4. An
emerging concept in molecular toxicology is that
reactive intermediates attack cysteine residues in
‘‘sensor trigger’’ proteins, producing structural
alterations and activating signaling networks that alter
patterns of gene expression or commit cells to cell
death/ cell survival decisions. A classic example of
such a trigger protein is KEAP1, a thiol-rich species
that upon oxidative damage releases its partner protein
NRF2, allowing the latter to activate various genes
involved in antioxidant defense and conjugative drug
metabolism (Dinkova-Kostova et al., 2002). In contrast,
little is known concerning the transcriptional
activation of Mrp4, although the recent finding that
Mrp4 expression in the basolateral membranes of murine
hepatocytes is regulated by constitutive androstane
receptor suggests future work could explore the
interaction of reactive intermediates with this nuclear
receptor and its partner proteins (Assem et al., 2004).
Finally, by providing new insights into the range of
molecular alterations occurring in the
xenobiotic-challenged rodent liver, the new findings
underscore inadequacies in existing classification
systems commonly used in toxicology. While the
‘‘toxicokinetic versus toxicodynamic’’ categorization is
useful in conceptual terms, the findings of Aleksunes
and associates demonstrate that such binary
classifications may not fully embrace all of the
molecular events occurring in xenobioticchallenged
tissues. Thus, in addition to the toxicokinetic
alterations discussed above (i.e., induced membrane
transporter expression), strong hepatic induction of the
stress protein HO-1 was detected in acetaminophen- and
CCl4-intoxicated mice. The latter response clearly
belongs to the category of toxicodynamic alterations,
since HO-1 is an inducible stress response protein that
catabolizes heme groups released from drug-metabolizing
enzymes after damage by oxidants and electrophiles
(Bauer and Bauer, 2002). By confirming that adaptive
responses to chemical hepatotoxicants includes molecular
changes that encompass features of both toxicokinetic
and toxicodynamic alterations, the work by Aleksunes et
al. (2005 and manuscript in this journal) underscores
the valuable contributions of mechanistic studies in
toxicology.
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