**4. Redox alterations in hepatotoxicity**

The role played by the cellular redox state in the hepatic pathophysiological mechanisms is not well defined. Indeed, hepatotoxicity has been much linked to the oxidative status and deficiencies in the liver antioxidant system. For instance, in the case of the alcoholic liver disease (ALD) mainly represented by a chronic stage of alcoholic steatohepatitis (ASH), alter‐ ations in the cell redox state have been implicated in injured hepatocytes [40]. Ethanol is metabolized via alcohol dehydrogenase (ADH), the microsomal ethanol oxidizing system, and by catalase in the liver peroxisomes, although the activity of ADH is responsible for most of the ethanol catabolism. In this pathway, NAD+ is reduced by a transfer of hydrogen (and one electron, e**<sup>−</sup>** ) to NADH, with the concomitant production of acetaldehyde. The NADP+ can be also reduced, and hydrogen equivalents from ethanol, but not NADH, are transferred from the cytosol to the mitochondria via a shuttle mechanism such as the malate shuttle, the fatty acid elongation cycle, and/or the α‐glycerophosphate cycle; therefore, mitochondria become more reduced [41, 42].

Lately, the nonalcoholic steatohepatitis (NASH) is becoming a pathological entity gaining a significant public health concern [43]. NASH is a progressive form of nonalcoholic fatty liver disease (NAFLD) and features of NASH include steatosis, inflammation and varying degrees of fibrosis, and seems to follow a 2‐hit model, where the "1st Hit" involves excess of lipid accumulation in the liver, which sensitizes the liver to the "2nd Hit". This "2nd Hit" involves inflammation, oxidative stress, liver damage and fibrosis [44]. Here, multiple cellu‐ lar processes play important roles in maintaining the NAD+ /NADH ratio. For example, dur‐ ing glycolysis, β‐oxidation, and the TCA activity, NAD+ is reduced to NADH. Within the mitochondria, NADH is oxidized by the electron transport chain enzymes during oxidative phosphorylation.

Laboratory animals exposed to high fat diet exhibit impaired oxidative metabolism through reduced electron transport chain activity [45]. Then, it is possible that those animals exposed to high fat during late gestation and early postnatal life present a state of redox imbalance, and although their livers are able to readily reduce NADH during catabolic redox reactions (in response to increased fat intake), the ability to replenish NAD+ reserves is reduced due to impaired oxidative capacity [46]. High fat feeding is associated with depleted NAD+ reserves and reduced SIRTs abundance, both established hallmarks of metabolic aging, and supple‐ mentation with factors reversing the effects of depleted NAD+ reserves, and/or SIRT1 and SIRT3 abundance rescue the increased susceptibility to develop severe fatty liver disease in the adult life [47].

Liver steatosis induces a reduced state in cytosol and mitochondria of hepatocytes, as dem‐ onstrated by alterations in the NADH/NAD+ ratio calculated from the β‐hydroxybutyrate dehydrogenase and lactate dehydrogenase reactions [48]. The increased formation of reduc‐ ing equivalents could impair fatty acids oxidation and the TCA cycle [49], but it could also enhance the formation of glycerol‐3‐phosphate and thus lipogenesis [50]. Saturation of lipids may also modify cellular redox status. Among free fatty acids, monounsaturated fatty acids, such as oleic acids, are less toxic than palmitate, a saturated acid, because the latter increases the NADH/NAD+ ratio and promotes uncoupling between glycolysis and TCA cycle fluxes, leading to increased ROS production [51]. The hepatic accumulation of saturated fatty acids can promote redox imbalance and the formation of reactive oxygen intermediates, mainly inducing endoplasmic reticulum (ER) stress and apoptosis [52].

**4. Redox alterations in hepatotoxicity**

164 Redox - Principles and Advanced Applications

of the ethanol catabolism. In this pathway, NAD+

lar processes play important roles in maintaining the NAD+

(in response to increased fat intake), the ability to replenish NAD+

mentation with factors reversing the effects of depleted NAD+

onstrated by alterations in the NADH/NAD+

ing glycolysis, β‐oxidation, and the TCA activity, NAD+

one electron, e**<sup>−</sup>**

more reduced [41, 42].

phosphorylation.

the adult life [47].

The role played by the cellular redox state in the hepatic pathophysiological mechanisms is not well defined. Indeed, hepatotoxicity has been much linked to the oxidative status and deficiencies in the liver antioxidant system. For instance, in the case of the alcoholic liver disease (ALD) mainly represented by a chronic stage of alcoholic steatohepatitis (ASH), alter‐ ations in the cell redox state have been implicated in injured hepatocytes [40]. Ethanol is metabolized via alcohol dehydrogenase (ADH), the microsomal ethanol oxidizing system, and by catalase in the liver peroxisomes, although the activity of ADH is responsible for most

be also reduced, and hydrogen equivalents from ethanol, but not NADH, are transferred from the cytosol to the mitochondria via a shuttle mechanism such as the malate shuttle, the fatty acid elongation cycle, and/or the α‐glycerophosphate cycle; therefore, mitochondria become

Lately, the nonalcoholic steatohepatitis (NASH) is becoming a pathological entity gaining a significant public health concern [43]. NASH is a progressive form of nonalcoholic fatty liver disease (NAFLD) and features of NASH include steatosis, inflammation and varying degrees of fibrosis, and seems to follow a 2‐hit model, where the "1st Hit" involves excess of lipid accumulation in the liver, which sensitizes the liver to the "2nd Hit". This "2nd Hit" involves inflammation, oxidative stress, liver damage and fibrosis [44]. Here, multiple cellu‐

mitochondria, NADH is oxidized by the electron transport chain enzymes during oxidative

Laboratory animals exposed to high fat diet exhibit impaired oxidative metabolism through reduced electron transport chain activity [45]. Then, it is possible that those animals exposed to high fat during late gestation and early postnatal life present a state of redox imbalance, and although their livers are able to readily reduce NADH during catabolic redox reactions

and reduced SIRTs abundance, both established hallmarks of metabolic aging, and supple‐

SIRT3 abundance rescue the increased susceptibility to develop severe fatty liver disease in

Liver steatosis induces a reduced state in cytosol and mitochondria of hepatocytes, as dem‐

dehydrogenase and lactate dehydrogenase reactions [48]. The increased formation of reduc‐ ing equivalents could impair fatty acids oxidation and the TCA cycle [49], but it could also enhance the formation of glycerol‐3‐phosphate and thus lipogenesis [50]. Saturation of lipids may also modify cellular redox status. Among free fatty acids, monounsaturated fatty acids, such as oleic acids, are less toxic than palmitate, a saturated acid, because the latter increases

impaired oxidative capacity [46]. High fat feeding is associated with depleted NAD+

) to NADH, with the concomitant production of acetaldehyde. The NADP+

is reduced by a transfer of hydrogen (and

/NADH ratio. For example, dur‐

reserves is reduced due to

reserves, and/or SIRT1 and

ratio calculated from the β‐hydroxybutyrate

reserves

is reduced to NADH. Within the

can

Moreover, the pathogenesis of early‐stage NASH is characterized by hyperinsulinemia and *de novo* synthesis of fatty acids and nascent triacylglycerides, which are deposited as lipid drop‐ lets within the hepatocytes. Hyperinsulinemia shifts the energy supply from glucose to ketone bodies, and the high ketone body concentration induces the overexpression of cytochrome P450 2E1 (CYP2E1), resulting in unsaturated fatty acids peroxidation and aldehydes produc‐ tion [53]. The NADPH oxidase‐derived ROS from arachidonic acid peroxidation can induce the nuclear translocation of *EGR1*, which in turn can stimulate the expression of the downstream genes *ATF3* and *GADD45G*. ATF3 is a transcription factor involved in cell proliferation, apop‐ tosis, and invasion [54]. The increased redox signaling plays a central role in promoting insulin resistance in the liver in early NASH, followed by fibrogenesis through activation of protein kinase R (PKR), protein kinase R‐like endoplasmic reticulum kinase (PERK) key stress kinases [55]. There is evidence that activation of the purinergic receptor P2X7 can give rise to NADPH oxidase activation, leading to Kupffer cell activation, a key event in NASH progression [56].

Peroxisomal oxidation of fatty acids is the normal route of metabolism of very long chain fatty acids and dicarboxylic acids, where electrons from FADH2 and NADH are transferred directly to O2 [57]. Moreover, fatty acids not oxidized by mitochondria are mainly oxidized by CYP2E1; a process that further increases ROS production [58]. Therefore, many cellular systems are important sources of ROS, including the mitochondrial respiratory chain [59], the cytochrome P450s [60], oxidative enzymes (xanthine oxidase, aldehyde oxidase, cyclooxy‐ genase, monoamine oxidase (MAO), and the NADPH oxidase complex) [61]. The uncoupling protein 2 (UCP‐2) may also enhance the reoxidation of NADH into NAD+ , which is required for both β‐oxidation and the TCA [62]; UCP‐2 oxidation might be considered an attempt to prevent steatosis by increasing hepatic fatty acid oxidation [63].

Moreover, it is accepted that in the NAFLD, depletion of hepatic antioxidants may contribute to the progression of steatosis to NASH by increasing oxidative stress that produces lipid peroxidation, inflammation, and fibrosis. Indeed, metabolic adaptations resulting from severe GSH deficiency seem to protect against the development of steatohepatitis [64]. In summary, all the redox alterations seem to be deeply implicated in the onset of NASH and in its progres‐ sion to liver fibrosis and a putative installation of a cirrhotic process.

However, a possible role of a disturbed cell redox state is much less known in cirrhosis. Nonetheless, it has been suggested that collagen metabolism could be influenced by changes in redox state. It has been postulated that conversion of glutamic acid to proline [65], a decreased NAD+ /NADH ratio [66], as well as impediment of proline transport and oxidation, could increase the liver proline pool, as a fundamental collagen component in the onset of liver fibrosis [67].

In experimental models of rat liver fibrosis/cirrhosis, mitochondrial function and structure show a variety of alterations. ATP synthesis is reduced in rats treated with CCl<sup>4</sup> or thioacet‐ amide, as well as in rats with secondary biliary cirrhosis. These alterations are compensated by increasing mitochondrial volume per hepatocyte and possible augmentation of extrahe‐ patic ATP production, as an effort for maintaining mitochondrial function in the cirrhotic liver [68]. These reports agree with the statement that in perfused cirrhotic livers, a reduced cytoplasmic and mitochondrial redox states occur accompanied by a diminished activity in the mitochondrial electron‐transport chain [69].
