NADPH depletion

Depletion of other cofactors such as UTP, NADH, and NADPH may also be involved in cell injury either directly or indirectly. Thus, the role of NADPH in maintaining reduced GSH levels means that excessive GSH oxidation such as caused by certain quinines, which undergo redox cycling, may in turn cause NADPH depletion (see below). Alternatively, NADPH may be oxidized if it donates electrons to the foreign compound directly. However, NADPH may be regenerated by inter conversion of NAD+ to NADP+. Some quinones such as menadione, l,2-dibromo-3-chloropropane (DBCP), and hydrogen peroxide also cause depletion of NAD, but probably by different mechanisms. Thus, with menadione, the depletion may be the result of... 

When the catalytic reaction of 6-hydroxymellein synthase is carried out in the absence of NADPH or with monomeric enzyme, keto-reduction of the carbonyl group of the triketomethylene chain does not take place, and the synthase liberates triacetic acid lactone instead of 6-hydroxymellein [64, 71]. However, the efficiencies of product formation are markedly lower than for the normal reaction. Two mechanisms could account for the low efficiency of triacetic acid lactone formation observed in the monomeric and the NADPH-depleted dimeric forms of 6-hydroxymellein synthase. These are 1) Reduced affinity of the cosubstrates acetyl-CoA and/or malonyl-CoA for the enzyme protein with the incomplete reaction centers 2) Reduced rate of reaction of acyl-CoA condensation and/or product liberation. To examine these possibilities, kinetic parameters of the two triacetic acid lactone-forming reactions were compared with those of the normal reaction which produces 6-hydroxymellein. The Km value of 6-hydroxymellein synthase for acetyl-CoA in the normal reaction was estimated to be 22 pM, while in both the NADPH-depleted dimer and the monomer reactions the affinity of 6-hydroxymellein synthase protein for acetyl-CoA was markedly lower at 284 and 318 pM respectively. By contrast the Km values for malonyl-CoA in the normal and the two abnormal reactions were essentially the same (40 - 43 pM), indicating that the affinity of 6-hydroxymellein... 

This mechanism is now considered to be of importance for the protection of LDL against oxidation stress, Chapter 25.) The antioxidant effect of ubiquinones on lipid peroxidation was first shown in 1980 [237]. In 1987 Solaini et al. [238] showed that the depletion of beef heart mitochondria from ubiquinone enhanced the iron adriamycin-initiated lipid peroxidation whereas the reincorporation of ubiquinone in mitochondria depressed lipid peroxidation. It was concluded that ubiquinone is able to protect mitochondria against the prooxidant effect of adriamycin. Inhibition of in vitro and in vivo liposomal, microsomal, and mitochondrial lipid peroxidation has also been shown in studies by Beyer [239] and Frei et al. [240]. Later on, it was suggested that ubihydroquinones inhibit lipid peroxidation only in cooperation with vitamin E [241]. However, simultaneous presence of ubihydroquinone and vitamin E apparently is not always necessary [242], although the synergistic interaction of these antioxidants may take place (see below). It has been shown that the enzymatic reduction of ubiquinones to ubihydroquinones is catalyzed by NADH-dependent plasma membrane reductase and NADPH-dependent cytosolic ubiquinone reductase [243,244]. 

Dihydrofolate reductase acts as an auxiliary enzyme for thymidylate synthase. It is involved in the regeneration of the coenzyme N, N -methylene-THF, initially reducing DHF to THF with NADPH as the reductant (see p. 418). The folic acid analogue methotrexate, a frequently used cytostatic agent, is an extremely effective competitive inhibitor of dihydrofolate reductase. It leads to the depletion of N, N -methylene-THF in the cells and thus to cessation of DNA synthesis. 

The outcome of oxidative stress is a depletion of cellular GSH, NADPH, NADH, and ATP, and also damage to lipid membranes, structural and enzymatic proteins, and DNA. 

If a large amount of the toxic substance is present, then the detoxication processes present are overwhelmed. Excess super oxide is produced, reduced GSH and NADPH are depleted, and hydroxyl radicals and singlet oxygen are formed. This is the condition known as oxidative stress (see also below this chapter). As well as causing lipid peroxidation, ROS will also cause DNA damage and damage to proteins. 

However, the reactive metabolite will cause other changes as well as binding to protein. Thus, NAPQI will react both chemically and enzymatically with GSH to form a conjugate and will also oxidize it to GSSG and in turn be reduced back to paracetamol. This cyclical process may explain the occurrence of extensive depletion of GSH. NADPH will also reduce NAPQI and in turn be oxidized to NADP, although reduction via GSH is probably preferential. NADPH oxidation may also result from GSSG reduction via GSH peroxidase (Fig. 7.18). 

The depletion of GSH and NADPH will allow the oxidation of protein sulfydryl groups, which may be an important step in the toxicity. Thus, GSH and protein sulfydryl groups, such as those on Ca2+-transporting proteins, are important for the maintenance of intracellular calcium homeostasis. Thus, paracetamol and NAPQI cause an increase in cytosolic calcium, and paracetamol inhibits the Na+/K+ ATPase pump in isolated hepatocytes. 

Lipid peroxidation/oxidative stress (Fig. 7.36). Lipid peroxidation products (e.g., malondialdehyde) have been detected, and GSH is depleted seemingly by oxidation rather than conjugation, and prior depletion of GSH increases the toxicity. NADPH is... 

Lipid peroxidation will oxidize GSH, and the reduction of this oxidized glutathione (GSSG), catalyzed by GSH reductase, will also use NADPH and hence contribute to the depletion of this nucleotide (Fig. 7.41). The hexose monophosphate shunt will be stimulated to... 

The result is redox cycling, which produces active oxygen species, which can deplete NADPH and GSH and potentially cause peroxidation of membrane lipids. NADPH is generated by the hexose monophosphate shunt, and NADP can also be reduced by GSH (Fig. 6.18). 

These drugs (e.g., cephaloridine) may be nephrotoxic causing proximal tubular necrosis. Cephaloridine is actively taken up from blood into proximal tubular cells by OAT 1. The drug therefore accumulates in the kidney. Metabolic activation via cytochrome P-450 may be involved. GSH is oxidized, and as NADPH is also depleted, the GSSG cannot be reduced back to GSH. As vitamin E-depleted animals are more susceptible, it has been suggested that lipid peroxidation may be involved. Damage to mitochondria also occurs. 

---