William F. Salminen

Correlation between glutathione oxidation and trimerization of heat shock factor 1, an early step in stress induction of the Hsp response.

The heat shock protein (Hsp) response is induced by heat shock and a large variety of different chemicals. Searching for a common denominator of these different inducers, we and others developed the notion that all inducers may generate abnormally folded, i.e. non-native, proteins, and that such non-native proteins may trigger the Hsp response. Experimentation prompted by this notion resulted, for example, in the demonstration that chemically denatured proteins, introduced in cells by microinjection, can activate the response. Based on the chemical nature of inducers and on results reported from several studies, we hypothesized that inducers of the Hsp response may be generally capable of triggering oxidation of non-protein thiols, particularly glutathione. Such oxidation is known to lead to formation of glutathione-protein mixed disulfides and protein-protein disulfides. Presumably, thiol adduction and cross-linking would affect the structure of proteins involved, resulting in unfolding of a fraction of these proteins, causing heat shock factor (Hsf) activation. To test the feasibility of this hypothesis, thirteen different inducers were selected, and it was shown that all chemical inducers as well as heat shock cause drastic oxidation of glutathione under conditions under which they induce HSE DNA-binding activity. Under the same conditions, all chemical inducers and heat shock also cause trimerization of Hsf1. For several inducers, it was also shown that they enhance thiol oxidation of proteins. Finally, in vitro experiments support the notion that activation of Hsf1 does not require oxidation of the factor itself or of its coregulators. These results are in complete agreement with the above hypothesis.

Identification of Urinary microRNA Profiles in Rats That May Diagnose Hepatotoxicity

Circulating microRNAs (miRNAs) have emerged as novel noninvasive biomarkers for several diseases and other types of tissue injury. This study tested the hypothesis that changes in the levels of urinary miRNAs correlate with liver injury induced by hepatotoxicants. Sprague-Dawley rats were administered acetaminophen (APAP) or carbon tetrachloride (CCl(4)) and one nonhepatotoxicant (penicillin/PCN). Urine samples were collected over a 24 h period after a single oral dose of APAP (1250 mg/kg), CCl(4) (2000 mg/kg), or PCN (2400 mg/kg). APAP and CCl(4) induced liver injury based upon increased serum alanine and aspartate aminotransferase levels and histopathological findings, including liver necrosis. APAP and CCl(4) both significantly increased the urinary levels of 44 and 28 miRNAs, respectively. In addition, 10 of the increased miRNAs were in common between APAP and CCl(4). In contrast, PCN caused a slight decrease of a different nonoverlapping set of urinary miRNAs. Cluster analysis revealed a distinct urinary miRNA pattern from the hepatotoxicant-treated groups when compared with vehicle controls and PCN. Analysis of hepatic miRNA levels suggested that the liver was the source of the increased urinary miRNAs after APAP exposure; however, the results from CCl(4) were equivocal. Computational analysis was used to predict target genes of the 10 shared hepatotoxicant-induced miRNAs. Liver gene expression profiling using whole genome microarrays identified eight putative miRNA target genes that were significantly altered in the liver of APAP- and CCl(4)-treated animals. In conclusion, the patterns of urinary miRNA may hold promise as biomarkers of hepatotoxicant-induced liver injury.

Potential of extracellular microRNAs as biomarkers of acetaminophen toxicity in children.

UNLABELLED Developing biomarkers for detecting acetaminophen (APAP) toxicity has been widely investigated. Recent studies of adults with APAP-induced liver injury have reported human serum microRNA-122 (miR-122) as a novel biomarker of APAP-induced liver injury. The goal of this study was to examine extracellular microRNAs (miRNAs) as potential biomarkers for APAP liver injury in children. Global levels of serum and urine miRNAs were examined in three pediatric subgroups: 1) healthy children (n=10), 2) hospitalized children receiving therapeutic doses of APAP (n=10) and 3) children hospitalized for APAP overdose (n=8). Out of 147 miRNAs detected in the APAP overdose group, eight showed significantly increased median levels in serum (miR-122, -375, -423-5p, -30d-5p, -125b-5p, -4732-5p, -204-5p, and -574-3p), compared to the other groups. Analysis of urine samples from the same patients had significantly increased median levels of four miRNAs (miR-375, -940, -9-3p and -302a) compared to the other groups. Importantly, correlation of peak serum APAP protein adduct levels (an indicator of the oxidation of APAP to the reactive metabolite N-acetyl-para-quinone imine) with peak miRNA levels showed that the highest correlation was observed for serum miR-122 (R=0.94; p<0.01) followed by miR-375 (R=0.70; p=0.05). CONCLUSION Our findings demonstrate that miRNAs are increased in children with APAP toxicity and correlate with APAP protein adducts, suggesting a potential role as biomarkers of APAP toxicity.

Green tea extract can potentiate acetaminophen-induced hepatotoxicity in mice

Green tea extract (GTE) has been advocated as a hepatoprotective compound and a possible therapeutic agent for acetaminophen (APAP) overdose. This study was conducted to determine if GTE can provide protection against APAP-induced hepatotoxicity. Three different exposure scenarios were tested. The first involved administering APAP (150 mg/kg, orally) to mice followed 6h later by GTE (500 or 1000 mg/kg). The other two involved administering GTE prior to the APAP dose. GTE (500 or 1000 mg/kg, orally) was administered 3h prior to APAP (200 mg/kg, orally) or for three consecutive days (once-daily) followed by APAP (300 mg/kg) on the fourth day. Indices of hepatotoxicity were assessed 24h after the APAP dose. GTE potentiated APAP-induced hepatotoxicity when administered after the APAP dose. GTE caused significant glutathione depletion and this effect likely contributed to the observed potentiation. In contrast, GTE provided protection against APAP-induced hepatotoxicity when administered prior to the APAP dose. GTE dramatically decreased APAP covalent binding to protein indicating that less reactive metabolite was available to cause hepatocellular injury. These results highlight the potential for drug-dietary supplement interactions and the importance of testing multiple exposure scenarios to adequately model different types of potential interactions.

Metabolomics evaluation of the effects of green tea extract on acetaminophen-induced hepatotoxicity in mice.

Green tea has been purported to have beneficial health effects including protective effects against oxidative stress. Acetaminophen (APAP) is a widely used analgesic drug that can cause acute liver injury in overdose situations. These studies explored the effects of green tea extract (GTE) on APAP-induced hepatotoxicity in liver tissue extracts using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance spectroscopy. Mice were orally administered GTE, APAP or GTE and APAP under three scenarios. APAP alone caused a high degree of hepatocyte necrosis associated with increases in serum transaminases and alterations in multiple metabolic pathways. The time of GTE oral administration relative to APAP either protected against or potentiated the APAP-induced hepatotoxicity. Dose dependent decreases in histopathology scores and serum transaminases were noted when GTE was administered prior to APAP; whereas, the opposite occurred when GTE was administered after APAP. Similarly, metabolites altered by APAP alone were less changed when GTE was given prior to APAP. Significantly altered pathways included fatty acid metabolism, glycerophospholipid metabolism, glutathione metabolism, and energy pathways. These studies demonstrate the complex interaction between GTE and APAP and the need to employ novel analytical strategies to understand the effects of dietary supplements on pharmaceutical compounds.

MicroRNA expression profiles distinguish the carcinogenic effects of riddelliine in rat liver

Pyrrolizidine alkaloids (PAs) are the most common plant constituents that poison livestock, wildlife and humans. Riddelliine is a prototype genotoxic PA and has been nominated to be classified as a reasonably anticipated human carcinogen by the US National Toxicology Program (NTP) in the 12th Report on Carcinogens. Riddelliines nomination is due to the high incidence of liver tumours that were observed in both mice and rats in the NTP tumourigenicity bioassay study. In this current study, we explored whether riddelliine treatment could alter microRNA (miRNA) expression in rat liver and whether the possible deregulation of miRNA was related to mutagenicity and carcinogenicity of riddelliine. Groups of six rats were administered riddelliine at a mutagenic dose of 1 mg/kg body weight or with control vehicle 5 days a week for 12 weeks. A group of six rats treated with aristolochic acid, a renal carcinogen, was used as a tissue-specific negative control. The animals were sacrificed 1 day after the last treatment and the livers were isolated for miRNA expression analysis using miRNA microarrays. miRNA expression was significantly altered by riddelliine treatment. Principal component analysis and hierarchical clustering analysis showed that the miRNA expression profiles were clearly classified into two groups, riddelliine treatment versus other samples. Forty-seven miRNAs were significantly dysregulated by riddelliine treatment, among which 38 were up-regulated and 9 were down-regulated. Functional analysis of these differentially expressed miRNAs by riddelliine revealed that these miRNAs were involved in liver carcinogenicity and toxicity, such as liver proliferation, liver necrosis/cell death, hepatocellular carcinoma, liver hepatomegaly, liver inflammation and liver fibrosis. These results suggest that miRNAs actively respond to a mutagenic dose of riddelliine and the pattern of miRNA expression has the potential to be used as a biomarker of genotoxicity and carcinogenicity for riddelliine and possibly other PAs.

Hepatic cytochrome P450s attenuate the cytotoxicity induced by leflunomide and its active metabolite A77 1726 in primary cultured rat hepatocytes

The Black Box Warning section of the U.S. drug label for leflunomide was recently updated to include stronger warnings about potential hepatotoxicity from this novel anti-arthritis drug. Because metabolic activation is a key mechanism for drug-induced hepatotoxicity, we examined whether leflunomide and its major metabolite, A77 1726, are cytotoxic to primary rat hepatocytes and whether their toxicity is modulated by hepatic cytochrome P450s (CYPs). As measured by lactate dehydrogenase leakage, time-dependent cytotoxicity was observed at 250-500 μM for leflunomide and 330-500 μM for A77 1726 within 20 h. Unexpectedly, three nonisoenzyme-specific CYP inhibitors, including SKF-525A, metyrapone, and 1-aminobenzotriazole, did not reduce but remarkably enhanced the cytotoxicity of leflunomide or A77 1726. SKF-525A pretreatment notably rendered hepatocytes susceptible to as low as 15 μM leflunomide or A77 1726. Three isoenzyme-specific CYP inhibitors including alpha-naphthoflavone, ticlopidine, and ketoconazole that mainly target CYP1A, CYP2B/2C, and CYP3A, respectively, also enhanced the cytotoxicity. A strong synergistic effect, similar to SKF-525A alone, was noted using a combination of all three of the isoenzyme-specific inhibitors. Hepatocytes pretreated with the CYP inducer dexamethasone for 24 h exhibited decreased cytotoxicity to leflunomide and A77 1726. At the concentrations tested, the CYP inhibitors and inducer showed no cytotoxicity. These data demonstrate that the parent forms of leflunomide and A77 1726 are more toxic to hepatocytes than their poorly characterized metabolites, indicating that the metabolic process of leflunomide is a detoxification step rather than an initiating event leading to toxicity.

Green tea epigallocatechin gallate binds to and inhibits respiratory complexes in swelling but not normal rat hepatic mitochondria.

Epigallocatechin gallate (EGCG), the major flavonoid in green tea, is consumed via tea products and dietary supplements, and has been tested in clinical trials. However, EGCG can cause hepatotoxicity in humans and animals by unknown mechanisms. Here EGCG effects on rat liver mitochondria were examined. EGCG showed negligible effects on oxidative phosphorylation at 7.5-100μM in normal mitochondria. However, respiratory chain complexes (RCCs) were profoundly inhibited by EGCG in mitochondria undergoing Ca(2+) overload-induced mitochondrial permeability transition (MPT). As RCCs are located in mitochondrial inner membranes (IM) and matrix, it was reasoned that EGCG could not readily pass through IM to affect RCCs in normal mitochondria but may do so when IM integrity is compromised. This speculation was substantiated in three ways. (1) Purified EGCG-bound proteins were barely detectable in normal mitochondria and contained no RCCs as determined by Western blotting, but swelling mitochondria contained about 1.5-fold more EGCG-bound proteins which included four RCC subunits together with cyclophilin D that locates in mitochondrial matrix. (2) Swelling mitochondria consumed more EGCG than normal ones. (3) The MPT blocker cyclosporine A diminished the above-mentioned difference. Among four subunits of RCC II, only SDHA and SDHB which locate in mitochondrial matrix, but not SDHC or SDHD which insert into the IM, were found to be EGCG targets. Interestingly, EGCG promoted Ca(2+) overload-induced MPT only when moderate MPT already commenced. This study identified hepatic RCCs as targets for EGCG in swelling but not normal mitochondria, suggesting EGCG may trigger hepatotoxicity by worsening pre-existing mitochondria abnormalities.

Urinary Micrornas as Noninvasive Biomarkers for Acetaminophen- Induced Liver Injury

Several recent studies measured elevated levels of circulating plasma microRNAs (miRNAs) after toxicant-induced liver injury, most likely due to leakage from damaged hepatocytes. miRNAs have also been detected in urine with some of them being derived from organs outside of the urinary system, opening up their potential use as noninvasive biomarkers of disease or injury. Despite this potential, changes in urine miRNA profiles have not been investigated as biomarkers for drug-induced liver injury. In this study, urine miRNA profiles were assessed from rats treated with a single oral dose of acetaminophen (APAP: 100 or 1250 mg/kg). The low dose did not cause any clinical pathology or histopathological changes indicative of liver injury. In contrast, the high dose increased clinical pathology and histopathological indices of liver injury at 24 hours; however, there was a high inter-animal variability in these endpoints. No evidence of kidney injury was noted at either APAP dose. Urinary miRNA levels correlated best with the degree of liver centrilobular glycogen depletion. In contrast to the high inter-animal variability noted for serum alanine and aspartate aminotransferases and centrilobular liver necrosis, urinary miRNA levels were consistently elevated in all high dose animals. Alterations in the urinary miRNA profiles were also noted in the low dose animals, albeit fewer in number. These results suggest that specific miRNAs in the urine could reflect the severity of APAP-induced liver injury and therefore have the potential to be used as noninvasive preclinical and clinical biomarkers.

Current and emerging biomarkers of hepatotoxicity

Drug-induced liver injury (DILI) is of great concern to human health. Generally, liver function and injury is evaluated based upon clinical signs, a select group of serum clinical biomarkers, and occasionally liver biopsies. While alanine aminotransferase, the most commonly used biomarker of hepatocellular injury, is a sensitive marker of liver injury, it is not necessarily specific for liver injury. Furthermore, alanine aminotransferase levels may not always correlate with the extent of injury. Therefore, new hepatotoxicity biomarkers are needed that are more predictive and specific indicators of liver injury and altered function. In addition, no current biomarker provides prognostic information about ultimate outcome once injury occurs, and any new biomarker filling this need is desperately needed. The omics technologies, including genomics, proteomics, and metabolomics, are being used in preclinical animal studies as well as clinical studies to evaluate markers of hepatotoxicity in easily obtained biofluids, such as urine and serum. Recently, the evaluation of circulating microRNAs in urine and blood has also shown promise for the identification of novel, sensitive markers of liver injury. This review evaluates the current status of proposed biomarkers of hepatotoxicity from the omics platforms, as well as from analysis of microRNAs. A brief description of the qualification of proposed biomarkers is also given.