We applied an integrated approach to study PMCol-induced toxicity by evaluating the compound using classic toxicity endpoints, gene expression, and metabolomics of the liver, kidney, urine, and plasma. PMCol induced periportal hepatocellular hydropic degeneration, cytomegaly, and periportal hepatocellular fatty changes, clearly identifying the liver as the key target for PMCol toxicity.
Glutathione is the predominant intracellular reducing agent in liver. This tripeptide plays a significant role in phase II xenobiotic modification and prevents reactive oxygen species (ROS)–mediated oxidative deactivation of cellular proteins. Along with antioxidant enzymes (catalase and superoxide dismutase), glutathione maintains cellular redox homeostasis (Pastore et al., 2003
). Metabolomic evaluation of liver from PMCol-treated rats demonstrated depletion of both GSH and GSSG in the high-dose group and a concomitant decrease in plasma cys-glutathione disulfide, a marker of hepatic glutathione depletion. Depletion of glutathione also stimulates cysteine biosynthesis () through the trans-sulfuration pathway, specifically inducing the expression of cystathionine-γ-lyase (Kandil et al., 2010
). Dose-dependent decreases in methionine were found after 28 days of PMCol administration, corresponding with increases in both S-adenosylhomocysteine and cysteine ( and ). Furthermore, ophthalmate, a product of the glutathione synthetic pathway that is not redox reactive, was depleted in the high-dose group. This depletion in glutathione was associated with upregulation of the glutamate-cysteine ligase message and accumulation of the component amino acids, especially cysteine, glycine, and 2-aminobutyrate ()—suggesting inhibition of glutathione biosynthesis by PMCol or its metabolites in a manner analogous to buthionine sulfoximine, a known mechanism-based inhibitor of glutamate-cysteine ligase (gamma glutamylcysteine synthetase) (Griffith and Meister, 1979
Cascade of events and responses following daily PMCol administration.
In addition to depletion of liver glutathione, PMCol appeared to compromise hepatic function as evidenced by alterations in sulfur and polyamine metabolism. Metabolomics showed decreases in liver organic sulfates and increases in urinary β-alanine, taurine, and creatine in the high-dose groups; however, the above changes may indirectly be related to increased cysteine production resulting from decreased hepatic glutathione.
PMCol caused oxidative stress as evidenced by appreciable decreases in the cofactor FAD in hepatic tissue after 28 days of treatment, with an increase in the cofactor NAD(P)+ after 7 days (). The increase in NAD(P)+ was associated with an increase in the message for Nqo1, an enzyme that converts NAD(P)H to NAD(P)+ in the presence of reactive quinones (Nebert et al., 2002
; Ross et al., 2000
). Nqo1 is responsible for conversion of tocopherol quinone, generated from vitamin E chromanol ring interaction with lipid radicals to its hydroquinone (Siegel et al., 1997
). Given that PMCol possesses the same chromanol moiety as vitamin E, it may also be converted by lipid peroxy radicals to a reactive quinone and lead to generation of more ROS. Unlike vitamin E, PMCol is more water soluble, less membrane bound, and has more access to the cytosolic glutathione pool. An in vitro
distribution study demonstrated that after incubation with erythrocytes, PMCol, unlike α-tocopherol, was undetectable in membrane fractions (Koga et al., 1998
). Thus, owing to its higher water solubility, PMCol may interact with and deplete cytosolic liver glutathione by both conjugative and nonconjugative mechanisms.
Nqo1 has also been reported to be upregulated in the livers of rats exposed to acetaminophen, carbon tetrachloride, and bromobenzene all of which cause hepatic oxidative stress (Aleksunes et al., 2005
; Heijne et al., 2004
). Upregulation of Nqo1 during hepatic oxidative stress and damage is an adaptive and protective response to limit progression of liver damage by detoxifying ROS (Aleksunes et al., 2006
). Although Nqo1 can convert vitamin E quinone to a hydroquinone, which is known to scavenge superoxide and protect cells from oxidative damage, in our study upregulation of Nqo1 did not appear to protect against liver injury. This may be due in part to a decreased pool of vitamin E in treated rat livers. Our metabolomic data showed that the liver vitamin E pool was significantly decreased even though vitamin E was significantly increased in the plasma of rats treated with low and high doses of PMCol. Given the structural similarity between PMCol and vitamin E, it is possible that PMCol results in depletion of liver vitamin E by inhibiting its liver absorption and/or interfering with its cell membrane incorporation, thereby, preventing the protective effect of Nqo1 and leading to cell damage.
Based on the metabolomic data, PMCol competes with vitamin E and campesterol (a dietary metabolite) for cellular uptake. Vitamin E, campesterol, and cholesterol are transported by one of the major cholesterol transporters, Niemann-Pick C1-like 1(Abuasal et al., 2010
; Narushima et al., 2008
; Takada and Suzuki, 2010
). Furthermore, major lipophilic and hydrophilic dietary antioxidants and ergothionine (a highly abundant fungal metabolite in erythrocytes) showed reduced hepatic abundance with high doses of PMCol (). The role of ergothionine as an antiapoptotic agent and the identification of its transporter, ETT, have only recently been elucidated (Paul and Snyder, 2010
). Oxidized ergothionine is a strong reducing agent and until intracellular glutathione is depleted, reduced ergothionine is functionally inert.
During lipid biosynthesis, the liver generates palmitate and cholesterol from excess citrate, producing cytosolic acetyl-coA, acetoacetyl-coA, and malonyl-coA. In this study, PMCol administration altered normal liver function, specifically lipid biosynthesis and remodeling. Inhibition of lipid anabolism by PMCol is evidenced by depletion of malonyl-CoA and decreases in squalene and hepatic cholesterol (). Furthermore, PMCol caused an increase in the level of hepatic acetylcarnitine, indicating inhibition of acetyl-coA incorporation into these pathways.
Hepatic XO, which carries out successive oxidation of hypoxanthine to allantoin and generates hydrogen peroxide at each step, may also play a role in the PMCol-induced hepatotoxicity. The lower abundance of each downstream metabolite (specifically xanthine, urate, and allantoin) in the XO pathway with no change in the initial substrate (hypoxanthine) suggests that PMCol decreases hepatic XO activity ().
Toxicogenomic results were consistent with metabolomic findings, indicating that PMCol-hepatotoxicity is related to glutathione depletion. Various genes in the glutathione metabolic pathway were significantly affected (upregulated or downregulated) by PMCol. Two previously described biomarkers of hepatotoxicity induced by a conjugation-based mechanism of glutathione depletion—aldo-keto reductase family 7 member A3 (Akr7a3) and glutathione S-transferase, pi 1 (Gstp1) (Gao et al., 2010
)—were upregulated in liver after PMCol administration.
The genomics data also indicated altered expression of the CYP genes; several CYPs were upregulated, whereas others were downregulated. Additionally, PMCol plasma levels were reduced on day 28 compared with day 7 suggesting that PMCol may induce CYPs responsible for its own metabolism, leading to generation of glutathione conjugated intermediates of PMCol metabolite(s). The in vitro
and in vivo
metabolism of PMCol has been reported (Gorman et al., 2009
), and the main metabolites formed by rats were the dehydrogenated-sulfate, oxidized glucuronide, dehyrogenated glucuronide, sulfate, and glucuronide conjugates. In the current study, these metabolites were observed, as well as several others. More than 30 different putative PMCol metabolites were detected by LC-MS in tissues, plasma, and urine from PMCol-treated rats (). PMCol was primarily oxidized or dehydrogenated (probable products of CYPs) and further metabolized to sulfate, glucuronide, or methylated conjugates. One metabolite found in liver and plasma is a mercapturate of an oxidized form of PMCol, suggesting that glutathione conjugation of a PMCol metabolite may be partially responsible for glutathione depletion.
Induction of CYPs causes oxidative stress by uncoupling oxidation of NADPH to generate hydrogen peroxide (Bondy and Naderi, 1994
). ROS may be formed by auto-oxidation of the oxycytochrome P450 complex, generating superoxide, which in turn is converted to hydrogen peroxide by superoxide dismutase and catalase. Induction of CYP1A1 by chemicals has also been shown to cause ROS formation and lead to oxidative stress (Knerr et al., 2006
), likely by metabolizing the membrane lipid arachidonic acid to stable biologically active epoxides (Diani-Moore et al., 2006
; Rifkind, 2006
), thereby causing excessive lipid peroxidation and ROS formation. In this study, PMCol upregulated CYP1A1 gene and the oxidative stress biomarker gene, Hmox1. Although Jackson et al. (2009)
suggested that PMCol does not induce CYPs in vitro
and drug-drug interaction with PMCol is minimal, the panel of CYPs used in their study only included CYP 1A2, 2B6, and 3A4. Toxicogenomic data from the current study supports their findings but also indicates that PMCol can upregulate transcription of various other CYPs.
Based on the metabolomic and toxicogenomic evaluations, changes in several metabolic products (particularly, methionine, cysteine, and glutathione) and genes (specifically, Akr7a3, Gstp1, and CYPs) in the glutathione metabolism pathway may be potential early markers of PMCol-induced hepatotoxicity. Additionally, the 17 genes differentially expressed by at least twofold in all PMCol-treated groups may serve, upon further investigation, as early markers of PMCol-induced hepatotoxicity.
In contrast to earlier published data (Lindeblad et al., 2010a
), we report minor renal histopathology findings and no PMCol-attributable clinical chemistry changes indicative of kidney damage. Metabolomics results were inconclusive with respect to PMCol-induced kidney injury, and microscopic examination of the kidney showed slight renal tubular regeneration in rats given low and high doses of PMCol.
In summary, administration of PMCol depletes total glutathione in part by inhibition of its biosynthesis as evidenced by alterations in cellular metabolites within glutathione biosynthesis pathway (). PMCol also alters hepatic function and hepatic xenobiotic transcriptional response which in turn may lead to bioactivation of PMCol to an intermediate that may generate ROS and further consumption of hepatic glutathione (). Depletion of glutathione ultimately yields an adaptive metabolic response with the consumption of hepatic dietary antioxidants, increased methionine to cysteine conversion, and increases NAD(P)+ production—a maladaptive response demonstrated by decreased normophysiology of the liver, hepatocellular damage and death.
Integration of data generated by standard toxicology endpoints with metabolomics and genomics details the impact of daily administration of high doses of PMCol. PMCol-induced liver damage and dysfunction is likely due to glutathione depletion from inhibition of synthesis, depletion of liver glutathione, and modification of other drug metabolism pathways. Understanding the function of upregulated genes and bioactivation of PMCol by CYPs may elucidate the mechanism of action and provide early markers of toxicity of vitamin E-like chemopreventive agents.