PRINCIPLES OF TOXICOLOGY - Biology East Borneo

66 BIOTRANSFORMATION: A BALANCE BETWEEN BIOACTIVATION AND DETOXIFICATIONCytochrome P450 is a collective term for a group of related hemoproteins, all with a molecularweight (MW) around 50,000 daltons, which as will be seen later, differ in their substrate selectivityand in their ability to be induced and inhibited by drugs and chemicals (Table 3.4).Cytochrome P450–catalyzed oxidations are categorized by the nature of the atom that is oxidized(see Figure 3.8). Subsequent to the oxidation, the oxygen atom from molecular oxygen may be retainedwithin the major fragment of the chemical or it may be eliminated by molecular rearrangement (e.g.,O and N dealkylations).Whatever the atom oxidized, or the name given to the reaction, the cytochrome P450–mediatedoxidation involves the same cyclic three-step series (Figure 3.9).Step 1. The xenobiotic [X] first binds to the cytochrome at a substrate binding site on the protein andalters the conformation sufficiently to enable the efficient transfer of electrons to the heme fromNADPH via a nearby (see Figure 3.6) flavoprotein, NADPH cytochrome P450 reductase. (Theactivity of this FAD- and FMN-containing flavoprotein is often determined experimentally usingexogenously added mitochondrial cytochrome c rather than microsomal cytochrome P450 as theelectron acceptor and so is often identified as NADPH cytochrome c reductase). The conformationalchange can sometimes be seen in vitro (in the absence of electron transfer) as an alteration of theheme from a low-spin to a high-spin state, which results in a blue shift in the absorbance maximumof the hemoprotein. The gain at 390 nm and loss at 420 nm, when seen by difference spectroscopy,is termed a type I binding spectrum (not to be confused with phase I metabolism).Step 2. The reduction of the heme iron from its normal ferric state to the ferrous state allows amolecule of oxygen (O–O) to bind (the binding of CO rather than oxygen to ferrous cytochromeP450 in the in vitro situation provides a characteristic absorbance maximum around 450 nm, whichgives this cytochrome its name).Step 3. The ternary complex of xenobiotic, cytochrome, and oxygen receives another electron, eitherthrough the same flavoprotein as before or through an alternative path involving a differentflavoprotein in which the electron is first passed through cytochrome b 5, another cytochromepresent in the endoplasmic reticulum (see Figure 3.6). This alternate pathway for the second electroncan also use NADH as the pyridine nucleotide electron donor. The addition of the second electronto the ternary complex results in a eventual splitting of the molecular oxygen, one atom of whichoxidizes the chemical, the other atom picking up protons to form water, returning ferric cytochromeP450 to repeat the cycle.Flavoprotein-catalyzed oxidations differ from cytochrome P450–catalyzed oxidations in mechanismand in substrate selectivity. For the flavoproteins (a 65,000-dalton protein containing only FAD), theenzyme forms an activated oxygen complex (“cocked gun”) and the addition of a metabolizablechemical discharges this, in the process of becoming oxidized. The electrophilic oxygenated speciesattacks nucleophilic centers. A wide range of chemicals can thus be metabolized by this flavoprotein;the important feature for metabolism being a heteroatom (nitrogen, sulfur) presenting a lone pair ofelectrons (Table 3.5).Some compounds are metabolized both by flavin-containing monooxygenases and cytochromeP450 but to different products. An example is dimethylaniline, which is metabolized to the N-oxideby the flavoprotein and is N-demethylated by cytochrome P450.Nonmicrosomal Oxidations in other subcellular organelles can be catalyzed by flavoproteins (e.g.,monoamine oxidase in mitochondria) or pyridine nucleotide linked dehydrogenases (e.g., alcohol andaldehyde dehydrogenases in cytoplasm).Dehydrogenase-catalyzed oxidations do not involve molecular oxygen. The oxidation of thechemicals or drugs occurs through electron transfer to a pyridine nucleotide, usually NAD + . Most ofthe dehydrogenases are cytoplasmic in location. The most noteworthy of this class of enzymes inhumans is the dehydrogenase responsible for the metabolism of ethanol. In contrast to the major