Discussion

In the present study, we investigated whether altered gene expression patterns that are suggestive of oxidative stress at a sub-toxic dose of APAP, when no apparent toxicity was detected using routine histopathological and clinical chemistry measurements, could be phenotypically anchored by using a panel of sensitive biomarkers for oxidative stress and oxidative DNA damage. Our results substantiate the previously reported gene expression profiling data (91) demonstrating that the sub-toxic

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dose of APAP does induce oxidative stress as demonstrated by the significant accumulation of nitrotyrosine protein adducts and 8-OH-dG DNA lesions and the reduction in GSH content at 6 hrs post-dosing. This confirms that gene expression signatures can potentially serve as predictive indicators of toxicity with increasing expression and exacerbation of a gene signature. Thus, the data support the potential role of gene expression profiling as a sensitive and biologically-relevant endpoint in toxicology.

APAP is a common over-the-counter medication used for its analgesic and antipyretic properties; however, it is also one of the leading causes of drug-induced liver failure (103). At pharmacological doses, APAP is metabolized by sulfation and glucuronidation, and to a lesser extent, by cytochrome CYP2E1 that produces a reactive metabolite, NAPQI, which is detoxified by conjugation with GSH (99, 104). APAP- induced hepatotoxicity occurs when GSH reserves are exhausted allowing covalent binding of NAPQI to critical cellular proteins as APAP-cysteine adducts (105), ultimately disrupting their cellular function, see Figure 2.6. Many of these covalently bound proteins are within the mitochondria (106) resulting in reduced respiration (107) and increased superoxide production (108). Superoxide either reacts with nitric oxide to produce peroxynitrite, which is responsible for protein nitration (109), or dismutates to hydrogen peroxide whereby it can oxidize cellular macromolecules. The presence of nitric oxide, which is induced by APAP (110), is thought to block propagation of lipid peroxidation (111). It has been postulated that loss of mitochondrial function and concomitant generation of oxidative stress are central to APAP-induced hepatotoxicity (112).

It is well recognized that the metabolic activation of APAP leading to GSH depletion is an important step in APAP-induced liver toxicity. As expected, an overtly toxic dose of APAP given to rats significantly reduced GSH content to 60% less than

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control at 6 hrs post-dosing. Most surprisingly, a sub-toxic dose also significantly reduced GSH, albeit to a lesser extent. These data support earlier gene expression studies for oxidant stress at sub-toxic dose although the

A number of studies have reported elevated levels of nitrotyrosine protein adducts that precede and accompany APAP-induced hepatotoxicity in mice (109, 113). In this study, only rats given a sub-toxic dose of APAP, not overtly toxic doses, had significantly elevated levels of nitrotyrosine protein adducts in liver compared to controls. These data support the presence of oxidant stress as indicated by earlier gene expression studies for a sub-toxic dose of APAP but demonstrates that the oxidant species formed in the presence of sub-toxic and overtly toxic doses of APAP in the rat are not identical. It has been shown that nitration of tyrosine residues is not limited to peroxynitrite exposure but can occur via peroxidase enzymes such as glutathione peroxidase (114) which is impaired during APAP toxicity. Alternatively, it has been demonstrated that APAP is highly effective at preventing tyrosine nitration by peroxynitrite (115, 116). Thus, in this study overtly toxic doses of APAP, unlike sub-toxic doses, may be able to out compete tyrosine for peroxynitrite which may explain the differences observed with nitrotyrosine levels between these two dosing groups. Moreover, this data would suggest that nitrotyrosine is not associated with APAP- induced hepatotoxicity in the rat; an observation that is in direct opposition to what has been observed in numerous studies with mice. This may well reflect a mechanistic difference in APAP metabolism between these two rodent species.

The generation of ROS by either APAP metabolism or resulting mitochondrial damage can lead to direct or indirect oxidative DNA damage. Immunohistochemical and mass spectrometry methods found a significant accumulation of the potentially mutagenic DNA lesion, 8-OH-dG, at sub-toxic and overtly toxic doses of APAP.

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Accumulation of 8-OH-dG lesions preceded the onset of hepatic injury as reported by ALT and histopathology. The formation of 8-OH-dG from APAP exposure potentially results from mitochondrial oxidant stress where both superoxide and peroxynitrite are produced and can either directly or indirectly oxidize guanines in DNA (35, 43).

Recently, the quantified expression of base excision DNA repair (BER) genes was shown to be a sensitive in vivo biomarker of chemical-induced oxidative stress (84). Moreover, because this pathway encompasses broad specificity and multiple routes of repair, it allows greater sensitivity in the ability to detect oxidative DNA damage. The measurement of multiple genes involved in the BER pathway by an RNase protection assay was able to detect up-regulation of gene expression that correlated with the onset of centrilobular hepatic necrosis in addition to the rise and fall of ALT. However, the assay was unable to detect significant increases of BER genes at a sub-toxic dose where genomic profiling generated an oxidative stress signature that consisted primarily of genes that are involved in protecting the cell from oxidative stress. It is known that the redox state of the cell is one of many mechanisms involved in activating transcription factors involved in regulating the expression of DNA repair genes (117). Thus, it may be that at sub-toxic doses of APAP the apparent increase in expression of anti-oxidants, such as metallothioneins, may be sufficient in maintaining a redox equilibrium.

The accumulation of apurinic/apyrimidinic (AP) sites can result from oxidative DNA damage through an intermediary step of BER pathway, enzymatic cleavage, and chemical depurination. The induction of BER pathway and, in particular, AP endonuclease gene by APAP was not, however, corroborated by the accumulation of AP sites by any of the APAP treatments examined. A lack of evidence for an increase in AP sites may be manifested in the limited dose and time regimens examined in this study. Since the development of APAP toxicity typically occurs within the first 6h of exposure,

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the detection of abasic sites would be limited if repair occurred rapidly. Alternatively, the repair pathway could involve another route whereby generation of an abasic site is obsolete. The dissociation between expression of BER genes and accumulation of AP sites is not an unusual phenomenon and has been observed with other chemical hepatotoxicants (84).

The role of lipid peroxidation in APAP-induced hepatotoxicity has been controversial (118-120). Mitochondrial dysfunction leads to both increased production of superoxide and formation of peroxynitrite that are both capable of initiating lipid peroxidation; however, biochemical studies have shown that nitric oxide can prevent the propagation of lipid peroxidation reactions (111). This is supported by the fact that inhibiting nitric oxide production during APAP exposure leads to enhanced lipid peroxidation (120). Our studies revealed that lipid peroxidation, as measured by the presence of malondialdehyde, was not observed to be significant for any doses or times examined. Despite the generation of ROS/RNS by APAP, our work does not support the role of lipid peroxidation as a mediator of APAP-induced hepatotoxicity.

It should be considered that alterations in gene expression that are potentially indicative of cellular injury with an adverse outcome but are unsubstantiated by classical measures of toxicity may be a mere reflection of the tissue’s capacity to cope. As demonstrated in this study, an APAP-induced oxidant signature generated by a sub-toxic dose was corroborated using sensitive biomarkers for oxidative stress and DNA damage. However, the presence of genes having an anti-oxidant role within this signature may explain the lack of observable toxicity by classical measures. In order to discern whether gene expression alterations in critical cellular pathways represent benign homeostatic adjustments, indications of the potential for adverse effects or in fact represent adverse effects, especially at doses and times with no observable toxicity, will

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require the application of more sophisticated and sensitive tools that provide a mechanistic link between a chemical and the observed toxic effect.

In general, the acceptance of microarray expression data as a relevant endpoint in toxicological studies requires careful interpretation and validation. It has been suggested that this should be achieved using classical toxicological endpoints such as histopathology and clinical chemistry (121). One of the great promises of toxicogenomics is that it will be able to detect and predict toxicity at much earlier stages compared to existing methods; however, restricting validation of expression data to only classical endpoints, with their inherent lack of sensitivity, would bring the advancement of toxicogenomics as well as toxicology to an impasse. Most importantly, the enhanced sensitivity of microarray studies to detect subtle, early alterations in vital cellular pathways that may be indicative of adverse effects, but display no observable toxicity by conventional measures, can have serious ramifications in policy and regulatory decision making.

In summary, we show that incipient signs of oxidative stress can be observed with a sub-toxic dose of APAP based on the significant accumulation of both nitrotyrosine protein adducts and 8-OH-dG DNA lesions, markers anchored on the mechanism of APAP-induced liver toxicity. The use of sensitive biomarkers of oxidative stress and oxidative DNA damage revealed not only that mechanistic differences may exist in APAP metabolism between sub-toxic and overtly toxic doses in rats but also among rodent species. Gene expression profiling is a sensitive tool capable of detecting subtle cellular disturbances at doses and times unobtainable by classical toxicological measures. Thus, it has the potential to serve an essential role in predicative toxicology by generating gene signatures as biomarkers of incipient toxicity.

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Reduced glutathione concentration in rat liver following acetaminophen treatment µmoles rGSH/g liver

Time Control 150 mg/kg 1500 mg/kg 6h 5.5 ± 0.43 3.8 ± 0.13* 2.3 ± 0.15*

24h 5.2 ± 0.68 5.9 ± 0.02 4.6 ± 0.75

Rats were administered a single acute dose of acetaminophen by gavage at sub- toxic (150 mg/kg) or overtly toxic (1500 mg/kg) doses in 0.5% aqueous methyl cellulose (vehicle control). Liver tissue collected at 6 or 24h post-dosing was analyzed for reduced glutathione (rGSH) content as described in Materials and Methods. Hepatic rGSH concentration is expressed as µmoles/g liver ± standard deviation from 3 animals per group. *Statistical difference (p < 0.01) from control group using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test.

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Expression of DNA repair genes in rat liver after treatment with an overtly toxic dose (1500 mg/kg) of acetaminophen

DNA Repair Gene Control 6h 24h 48h

Ogg1, 8-oxoguanine DNA

glycosylase 1 3.8 ± 0.8 3.9 ± 0.8 8.2 ± 2.8* 10.2 ± 1.5*

Mpg, N-methylpurine DNA

glycosylase 6.0 ± 0.4 5.2 ± 1.3 4.8 ± 2.7 4.0 ± 0.8

Ape, purinic/apyrimidinic

endonuclease 1 23.5 ± 1.4 21.6 ± 5.2 34.8 ± 2.8* 30.8 ± 1.7

Pol β, polymerase (DNA

directed) β 13.2 ± 0.9 11.9 ± 1.3 18.7 ± 1.8* 15.8 ± 1.2

Pol δ, polymerase (DNA

directed) δ 1.5 ± 0.3 1.7 ± 1.7 6.0 ± 1.6* 8.8 ± 2.8*

Pcna, proliferating cell

nuclear antigen 20.7 ± 2.1 22.5 ± 2.6 33.6 ± 7.9 50.9 ± 7.6*

Parp, poly (ADP-ribose)

polymerase 15.3 ± 0.5 21.4 ± 1.3* 27.6 ± 3.8* 29.1 ± 1.6*

Mgmt, O6-methylguanine

DNA ethyltransferase 29.8 ± 1.3 24.1 ± 5.7 32.2 ± 6.6 32.1 ± 0.1

Total RNA was isolated from liver samples and analyzed by RNase protection assay. The results are mean ± standard deviation from 3 animals per group. The relative expression of each gene was normalized to the expression of the housekeeping gene L32. The control is pooled RNA from three biological replicates and then 6, 24, and 48h time points averaged together. The results from animals given a sub-toxic dose of APAP (150 mg/kg) are not presented since they were not significantly different from controls. *Statistical difference (p < 0.05) from control group using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test.

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A Sub-toxic dose of acetaminophen significantly increases nitro-tyrosine protein adducts in rat liver

Representative micrographs (200×) of liver tissue from rats immunostained for nitrotyrosine after treatment with (A) methyl cellulose control or (B) 150 mg/kg acetaminophen at 6, 24, and 48hrs post-dosing and (C) 1500 mg/kg and (D) 2000 mg/kg acetaminophen at 6 hrs post-dosing*. Representative micrograph (40×) of liver tissue from rats after treatment with 150 mg/kg acetaminophen demonstrating centralobular localization of nitrotyrosine staining (D). Immunostained liver sections for control (□) and 150 mg/kg (■) APAP were quantified by averaging percent area stained to total area at 200× in pericentral regions (F). Data are presented as mean + SD, n = 3 biological replicates per group. Data significantly different from control, p < 0.01, is denoted by asterisk. *Analysis of overtly toxic doses of APAP at 24 and 48 hrs post-dosing was not performed due to the extensive presence of necrotic tissue that often stains non- specifically. CV, central vein; PV, portal vein.

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Rat liver genomic DNA significantly accumulates 8-OH-dG adducts after 6 hrs treatment with sub-toxic and overtly toxic doses of acetaminophen

Representative micrographs (200×) of liver tissue immunostained for 8-OH-dG from rats treated with (A) methyl cellulose control, (B) 150 mg/kg, or (C) 1500 mg/kg acetaminophen 6 hrs post-dosing. Representative micrograph (40×) of liver tissue from rats after treatment with 150 mg/kg acetaminophen demonstrating centralobular localization of 8-OH-dG adducts (D). Immuno-stained liver sections for control (□), 150 mg/kg (■), or 1500 mg/kg (■) APAP were quantified by averaging percent nuclei stained to total nuclei within pericentral regions at 200× (E). Data are presented as mean + SD, n = 3 biological replicates per group. Data significantly different from control, p < 0.05, is denoted by asterisk. ND = not determined. CV, central vein; PV, portal vein.

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A sub-toxic dose of APAP significantly accumulates 8-OH-dG DNA adducts in rat liver as measured by capillary LC-MS/MS

(A) Standard calibration curve for 8-OH-dG by capillary LC-MS/MS. The ratio of the peak areas of 8-OH-dG (AS) to 275.5 fmoles [15_N

5]-8-OH-dG (AI) as the internal

standard plotted against the amount of 8-OH-dG ranging from 5.5 to 228 fmoles/µl. (B) Quantitative measure of 8-OH-dG DNA adducts in rat liver from control (□), 150 mg/kg (■), or 1500 mg/kg (■) APAP. Data are presented as mean ± SD from 3 animals per group. *Statistical difference (p < 0.05) from control group using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test.

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Acetaminophen has no effect on the accumulation of apurinic/apyrimidinic (AP) sites in rat liver

The number of AP sites in genomic DNA isolated from livers of control (□) and 150 mg/kg (■), or 1500 mg/kg (■) APAP at 6, 24, and 48h. The control is pooled RNA from three biological replicates and then 6, 24, and 48h time points averaged together. Data is given as mean + SD, n = 3. Statistical analysis by one-factor ANOVA (p < 0.05) found no significant difference between acetaminophen treated groups and control.

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Acetaminophen does not promote lipid peroxidation in rat liver

Representative micrographs (200×) of liver tissue immunostained for malondialdehyde from rats treated with (A) methyl cellulose control, (B) 150 mg/kg, or (C) 1500 mg/kg acetaminophen 6 hrs post-dosing. Immunostained liver sections for control (□), 150 mg/kg (■), or 1500 mg/kg (■) APAP were quantified by averaging percent area stained to total area at 200× in pericentral regions (D). Data are presented as mean + SD, n = 3 biological replicates per group. Statistical analysis by one-factor ANOVA (p < 0.05) found no significant difference between acetaminophen treated groups and control. ND = not determined.

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Phenotypic anchors of gene expression profiling for oxidative stress are a reflection of the proposed mechanism of APAP-induced hepatotoxicity

APAP is metabolized by cytochrome P450s to a reactive metabolite, N-acetyl-p- benzoquinone imine (NAPQI), which depletes glutathione (GSH) and covalently binds to cellular proteins as APAP-cysteine adducts. Mitochondrial injury leads to increased production and release of reactive oxygen and nitrogen species that promotes oxidative stress and DNA damage. The depletion of GSH and coupling reaction of superoxide (O2·-

) and nitric oxide (NO·) leads to formation of peroxynitrite (ONOO-_{) that reacts with}

protein tyrosine residues. It has been proposed that the production of NO·, induced by APAP, terminates lipid peroxidation propagation. The concomitant loss of mitochondrial function and generation of oxidative stress are postulated to have a central role in APAP-induced hepatoxicity. The sequence of events involved in APAP-induced hepatatoxicity are shown in bold, whereas the markers of oxidative stress and oxidative DNA damage measured in this study are shown in italics. ∗, markers that anchored gene expression signature suggestive of oxidative stress with a sub-toxic dose of APAP. The scheme is a summation of previously published reports on APAP-induced hepatotoxicity, see Discussion.

TEMPORAL CORRELATION OF PATHOLOGY AND DNA DAMAGE WITH GENE EXPRESSION IN A CHOLINE-DEFICIENT MODEL OF RAT LIVER INJURY

The text of this chapter is reproduced with permission from Hepatology 42(5): 1137-1147 (2005)

© 2005

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Hepatocellular carcinoma (HCC) is the terminal event in chronic liver diseases that include repeated cycles of cellular injury and regeneration. While much is known about the cellular pathogenesis and etiologic agents leading to HCC, the molecular events are not well understood. The choline-deficient (CD) model of rodent HCC involves the consecutive emergence of a fatty liver, apoptosis, compensatory proliferation, fibrosis, and cirrhosis that is markedly similar to the sequence of events typified by human HCC. Moreover, oxidative stress is thought to play a pivotal role in the progression of the disease. Here, we hypothesize that gene expression profiling can temporally mirror the histopathology and oxidative DNA damage observed with this model. We show that clusters of highly co-regulated genes representing distinct cellular pathways for lipid biosynthesis and metabolism, apoptosis, cell proliferation and tissue remodeling temporally correlate with the well-defined sequential emergence of pathological alterations in the progression of liver disease. Additionally, an oxidative stress signature was observed which was corroborated in a time-dependent manner with increases in oxidized purines and abasic sites in DNA. Collectively, expression patterns were strongly driven by pathology demonstrating that patterns of gene expression in advanced stages of liver disease are primarily driven by histopathological changes and to a much lesser degree by the original etiological agent. In conclusion, gene expression profiling coupled with the CD model of HCC provides a unique opportunity to unveil the molecular events associated with various stages of liver injury and carcinogenesis and to distinguish between causal and consecutive changes.

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The choline deficient (CD) diet is an extensively studied non-chemical induced, non-genotoxic model of rodent hepatocellular carcinoma (HCC) that produces a well- defined temporal pattern of pathological changes (Figure 3.1). It is characterized by an initial increase in triglycerides resulting in macro-vesicular fat deposition (steatosis) that quickly diffuses throughout the entire liver within 4 to 5 days (122). By 4 weeks, increased fat storage and oxidative stress are thought to contribute to hepatocellular injury which prompts apoptosis (123) and is coupled with compensatory liver regeneration (124). Fibrosis develops as activated hepatic stellate cells increase collagen production disrupting liver architecture, and eventually leads to cirrhosis by 30 weeks of treatment (125). Lastly, carcinomas develop with a 100% incidence by 52 weeks (126). The sequence of pathological events is remarkably similar to the progression of human HCC associated with hepatitis B (HBV) and C (HCV) viral infections, non-alcoholic fatty liver disease, and alcohol abuse.

The development and progression of HCC in the CD model is not well understood due to the complexity of genetic and epigenetic events that occur in