TABLE 5.—Admission rates (per 100 infants) by diagnosis, birth weight, and maternal smoking. Birth weight (g) Total <2,999 3,000-3,499 3,500+ (including unknown) 8 NS 8 NS 8 NS s NS (297) (2,826) (415) (4,098) (264) (3,195) (986) (9,686) Diagnosis Bronchitis and pneumonia 19.2 123 9.6 82 12.1 9.0 13.1 9.5 All other 26 19.9 145 14.6 15.2 13.3 16.9 15.5 Total 418 32.2 2.1 22.8 27.3 22.3 30.0 Aad NOTE. — S=Smokers; NS = Nonsmokers. Absolute numbers in parentheses. SOURCE: Harlap and Davies (42). which may exist between smoking and factors such as parental neglect or socioeconomic class. In addition, hospital admission rates may not be an accurate index of infant morbidity. Colley, et al. (22) and Leeder, et al. (54) studied the incidence of pneumonia and bronchitis in 2,205 children over the first 5 years of life in relation to the smoking habits of both parents. They found that a relationship between parental smoking habits and respiratory infection - in children occurred only during the first year of life (Table 6). They also showed a relationship between parental cough and phlegm production and infant infection (Table 6) which was found to be independent of the effect of parental smoking habits. The relationship between parental smoking and infant infection was greater when both parents smoked and increased with increasing number of cigarettes smoked per day. The relationship persisted after controlling for social class and birth weight. Thus, respiratory infections during the first year of life are related to parental smoking habits independently of parental symptoms, social class, and birth weight. Because of the dose-response relationship between parental smoking and infant respiratory infection established by Colley, et al. (22), it is reasonable to suspect that cigarette smoke in the atmosphere of the home may be the cause of these infections; however, other factors such as parental neglect may also play a role. Summary 1. Tobacco smoke can be a significant source of atmospheric pollution in enclosed areas. Occasionally, under conditions of heavy smoking and poor ventilation, the maximum limit for an 8-hour work exposure to carbon monoxide (50 ppm) may be exceeded. The upper limit for CO in ambient air (9 ppm) may be exceeded even in cases where ventilation is adequate. For an individual located close to a cigarette that is being smoked by someone else, the pollution exposure 11—33 TABLE 6.—Pneumonia and bronchitis in the first 5 years of life, by parents’ smoking habit and morning phlegm. Annual incidence of pneumonia and bronchitis per 100 children (Absolute numbers in parentheses) Both ex-smokers Year of followup Both nonsmokers One smoker Both smokers or one examoker All or smoking habit changed N O/B N 0/B N 0/B N 0/B N O/B 1 76 10.3 10.4 148 15.3 2.0 8.2 13.2 10.1 16.7 (343) (29) (424) (128) (888) (139) (546) (129) (1,652) (425) 2 81 8.3 Tl 15.5 87 9.2 65 10.7 TA 113 (322) (86) «= (365) (129) (286) (182) (599) (159) (1,572) (478) 3 69 8.1 10.5 94 79 11.0 8.2 11.6 8.4 10.6 (305) (37) (853) (107) (242) (154) (661) (173) (1,561) (471) 4 8.0 Wl 15 10.8 7.6 11.6 8.2 9.1 19 10.3 (287) (36) + (806) ~=«(102) (286) = (121) (685) (187) (1,524) (448) 67 14.7 5.6 9.4 3.9 10.6 64 13 59 9.1 (285) (34)-—«(267)—«(107)— (208) (482) (787) (219) (1,497) (492) on NOTE.—N =neither with winter morning phlegm; O/B ~ “active oxygen” 3. “Active oxygen” + drug — oxidized drug + A + H20 In sum: NADPH + Oz+ drug = NADP*++ He2+ oxidized drug. Key enzymes in the overall reactions are nicotinamide-adenine dinucleotide phosphate reductase (NADPH)-cytochrome C reductase, the flavin enzyme involved in the oxidation of NADPH, cytochrome P- 450, which in its reduced form is generally considered to be A, and NADPH cytochrome P-450 reductase, which functions in the reduction of oxidized cytochrome P-450. This mechanism requires that equivalent amounts of NADPH, oxygen, and substrate be utilized in the reaction. Stoichiometric relationships have been obtained for the hydroxylation of phenylala- nine by hepatic microsomes (26) and the hydroxylation of 17-hydroxy- progesterone by adrenal microsomes (8). Trimethylamine has been reported to stimulate NADPH oxidation by an amount equivalent to the amount of trimethylamine oxide formed (2), and hexobarbital was found to increase NADPH oxidation in accordance with stoichiometric expectations (62). However, in several studies (14, 15, 16, 17) Gillette and coworkers found that some drugs had no effect on NADPH 12—10 oxidation, whereas others had more of an effect than could be accounted for by the metabolism of the drug. Microsomes contain enzymes which oxidize NADPH and utilize molecular oxygen in the absence of drugs, greatly complicating the analysis. Whether or not a jrug stimulates or depresses NADPH oxidation would seem to depend upon whether or not it stimulates or depresses cytochrome P-450 reductase activity; this, in turn, would seem to depend upon whether the drug combines with cytochrome P-450 as a type I or as a type II sompound (17, 18, 19) as discussed below. Ernster and Orrenius (20) jemonstrated a 1:1:1 stoichiometry of oxygen utilization, NADPH disappearances, and formaldehyde formation from the oxidative demethylation. of aminopyrine. However, Estabrook and Cohen (1/1) found that stoichiometry did not support the basic assumption ofa mixed function oxidase reaction, that a mole of NADPH be oxidized for each mole of formaldehyde formed; two moles of nicotine-adenine dinucleotide phosphate (NADP) were formed per ‘mole of formalde- hyde, suggesting that the reaction is more complex than anticipated. Sasame, as cited in Mannering (37), did not find a stoichiometric relationship between NADPH and hexobarbital oxidation; the amount of NADPH oxidized was about 50 percent greater than the amount of hexobarbital metabolized. ; - Figure 1 shows the electron transfer system involving cytochrome P- 450 as conceived by Omura, et al. (43, 48). The first description of the microsomal system: responsible for drug metabolism (39, 40) included a role of nicotinamide-adenine dinucleo- tide reductase (NADH) as well as NADPH. From time to time since then, NADH has been implicated in reactions involving drug metabo- lism (6, 42, 62). Using the mechanism of peroxidase action as a model, Estabrook and Cohen (11) suggested a way in which NADH might contribute to the reaction (Figure 2). NADPH may serve as an electron donor, via a respiratory chain, direct to cytochrome P-450 with an associated branched pathway to cytochrome bs, the only cytochrome other than cytochrome P-450 found in microsomes. In this way, cytochrome bs might serve as a second electron donor to cytochrome P- 450 and thus satisfy the requirement of two electrons for the overall reaction. Sih and coworkers (57, 58) question the function of NADPH as solely to provide the reducing equivalents for cytochrome P-450 via the electron transfer system as shown in Figure 1. Mannering (35) discusses the three lines of evidence leading to the scheme given in Figure 8, which visualizes a dual role of NADPH in the oxidation of corticosteroids by mitochondria of the adrenal cortex. Much of the speculation regarding the components of the microsom- al drug metabolizing system existed because attempts to solubilize cytochrome P-450 in active form had failed, and it was necessary to employ crude microsomal preparations. In various studies (7, 31, 32, 33) 12—11 a rE Oo Oo + rE + oO 2 « 2 0 st a x ° 3 oO w x= oO xc 9 9 + 2 ot 7 + + © meee B+ u Oo aw +o 3 OQ Ow t o + z+ —_+ + I+ Zo Zo ir uw XK Fo Fp X NADP NADPH FIGURE 1.—Proposed electron transfer system employed in the microsomal metabolism of drugs. F,=flavoprotein (in the liver, cytochrome C reductase; in the adrenal, adrenodoxin reductase); NHIP = non-heme iron protein (in the adrenal, adrenodoxin) SOURCE: Omura, T. (43,48). Coon and Lu and their associates did much toward solving this problem. Solubilization of hepatic microsomes from the rabbit with a mixture of glycerol, dithiothreitol, and sodium deoxycholate in a potassium citrate buffer produced an extract which was resolved into a fraction 12—12 NADH NADPH { PIs | \ 7 mm cytbs, ' \ P-450- \ H20 SOH N ° n-B v QO nN ° Q N—-wo x a on ° Db + —__ COCO a FIGURE 2.—Scheme showing how NADH and cytochrome bs might contribute to the electron transfer system employed in the microsomal metabolism of drugs SOURCE: Estabrook, R. (11). containing cytochrome P-450, a fraction containing a NADPH reductase, and a fat soluble, heat stable fraction. All three fractions were necessary for the maximal oxidation of drugs (benzphetamine, aminopyrine, ethylmorphine, hexobarbital, norcodeine, p-nitroanisole) or for the w-hydroxylation of lurate. The criterion for the solubilization of cytochrome P-450 was that it remained in the supernatant fraction 12—13 NADP * NADPH noo Fe(!l)"Op ° 9 r_3- _~S- z-% z-§ . 4 a. a 9° N x + co a x Oo 9 x 2 c <é Ro Dd eo FF e-8-u ge a a Deen 2 AH o F = ef 8.5 7 t- iP a a a 8 = Ff 2 3 2 57. t P(oxid) FPired) F x NADPH NADPH FIGURE 3.—Scheme illustrating a proposed dual role of NADPH in the oxidation of corticosteroids by mitochondria on the adrenal cortex. FP = flavoprotein (adrenodoxin); NHIP = non-heme iron protein (adrenodoxin reductase) SOURCE: Sih, C. (87,58) of the preparation after centrifugation at 105,000 x g for 2 hours. These fractions may provide the opportunity for purification and identification of the components of the system. Both NADH and NADPH can act as the electron donor in the reduction of nitro compounds. The reaction is presumed to proceed to the primary amine through the formation of nitroso and hydroxyl- 12—14 H202 Hydroxylated Substrates O2 P-450-CO Reduced Product Qo 3 $2 3 a Oo = a eee o og So + a8 aa a 3 2 2 «ce ~ 6 8 ° 3 Zo Bs 23 $6 ve » og ° Be Sse 8 ZS « «8 eo 6d8 «& 6 < x NADPH NADP NADH NAD FIGURE 4.—Scheme showing how the microsomal electron transfer system might function in both the oxidation and reduction of drugs SOURCE: Gillette, J.R. (19). amine derivates. Nitroreductase is active only under anaerobic conditions. Sensitivity to oxygen may be due in part to the auto- oxidation of the hydroxylamine intermediate (19). In studies which employed p-nitrobenzoate as a substrate, Gillette, et al. (19) concluded that the reduction was mediated by cytochrome P-450. These investigators proposed an electron transport system which would explain both the oxidative and the reductive function of the microsomal drug-metabolizing system (Figure 4). 12—15 Components of the Microsomal Drug Metabolizing System Cytochrome P-450 Cytochrome P-450, earlier referred to as the CO-binding pigment, was first described by Klingenberg (29), Garfinkel (12), and Omura and Sato (44, 45, 46, 47). It is found in abundance not only in hepatic microsomes, but also in the microsomes and mitochondria from the adrenal cortex where it functions in the hydroxylation of steroids (11, 48), although not in the oxidation of most drugs. Lesser amounts are found in the kidney and intestinal mucosa (37). The presence of cytochrome P-450 has also been reported in mitochondria from the corpus luteum (67). Factors concerning cytochrome P-450 include (35): (1) its spectral characteristics; (2) its conversion to cytochrome P-420 by a wide variety of compounds, such as phospholipase A, sodium deoxycholate and urea; and (3) its concentration in hepatic microsomes, which is influenced by various drugs, varies with age and sex, and is reported to rise after fasting. Drugs and other foreign compounds bind to hepatic cytochrome P-450 to produce different spectra of two general types, type I and type II. Type I compounds give a different spectrum with a X max in the general range of 385-390 mp and \ min in the equally broad range of 418-427 mp; the A max and min given by type II compounds are 425-435 and 390-405 mp, respectively (54). Thus, with opposing A max and A min, type I and type II spectra are approximate mirror images of each other. Figure 5 presents type I (hexobarbital) and type II (aniline) spectra. Compounds that induce microsomal drug metabolism tend to be type I compounds, such as aminopyrine, 3,4 benzpyrene, coumarin, DDT, ethylmorphine, hexobarbital, and progesterone; one exception is nicotine, a type II compound, which is reported to be an inducing agent. Mannering (35) presents a thorough discussion of the signifi- cance of the binding of cytochrome P-450 to compounds. Cytochrome P1-450 (P-448, P-446, High Spin P-450, Type a P- 450) The mechanism by which phenobarbital and many other drugs stimulate the synthesis of the microsomal drug metabolizing system has long been considered to be different from the mechanism whereby PAHs produce their inductive effects (36). This early assumption was based on the knowledge that drugs such as phenobarbital induce the increased metabolism of a much larger number of drugs and other foreign substances than do the PAHs such as 3-methylcholanthrene (3- MC) or 3,4-benzpyrene (BP). Attempts to measure some of the differences between the two inductive processes led to the conclusion that PAHs cause the synthesis of a modified cytochrome P-450. For lack of a more suitable nomenclature for the microsomal hemoproteins, the hemoprotein cytochrome was named P1-450 (37, 55, 59, 60, 61). 12—16 S e222 7 eceo ° oO x em mM Te NO wo < TON QO ~e 2 xaooo 43 oO d g = 1s 2 ° 2 7* 0 x o 0 48 x w “ Oo 42 2 WM = o o € Of as = Mo x x Og:* 43 5 s 2&5 mo 9 Zz gce z Qoga 48 4 me oad vow a S36 < o Wig Ps 48 Dina ~ 9 2 Zz ly a < “ g irs] 42 re = ° z F223 s < i 8 xx 7s xes ~on7oO 80 oo-~* © 42 xempoow oO 1 1 1 LL. 1 l L o oO N = o = N o t 8 3 ° ° ° 3S ° 3° ° ° o O. o ° o Cw ‘O00 I g2