Genetic differences in drug metabolism associated with ocular toxicity.

The tissue localization and subcellular distribution of drug-metabolizing enzymes in the eye are described. With the use of inbred strains of mice, the [Ah] complex is shown to be an important experimental system for probing genetic differences in drug metabolism and related drug toxicities. Although the genetic system described in detail here involves mice, there is ample evidence that the same system operates in man. Genetic differences in acetaminophen- and naphthalene-induced cataract formation and and other ocular degeneration are shown to be related to the [Ah] complex. Because this toxicity appears similar to senile cataracts, we propose that certain types of drug-induced cataracts might exist among clinical populations of senile cataracts but that any cause-and-effect relationship would be very difficult to determine because of underlying interindividual differences in genetic predisposition. It is therefore suggested that genetic differences in drug metabolism be an important consideration in the clinical assessment of ocular toxicity caused by drugs and other environmental pollutants.


Introduction
Other contributions in this symposium are concerned with drug toxicity and pathology related to tissues of the head and neck. This paper deals more directly with the drug-metabolizing enzymes involved. These enzymes are responsible for toxification (i.e., potentiation of the toxic response by forming a reactive drug intermediate), as well as detoxication (metabolism leading to the excretion of innocuous products). This paper also emphasizes genetic differences in these enzymes, whereas genetics is not addressed in the other contributions. Although the genetic system described in detail here involves mice, there is ample evidence that the same system operates in man. It therefore should be obvious that, due to differences in genetic predisposition among individuals in the human population, a particular dose of an ophthalmic drug might be toxic to one person but not to another (and these *This paper was not presented at the Symposium on Target Organ Toxicity: Eye, Ear  April 1982 two persons might even be siblings). Similar genetic differences in toxicity caused by drugs and other environmental pollutants are expected to be manifest among other tissues of the body, as well as in the eye.
We first describe the tissue localization and subcellular distribution ofdrug-metabolizing enzymes in the eye. Next, the genetic system, called the [Ah] complex, is introduced. Finally, genetic differences in acetaminophen-and naphthalene-induced cataract formation (lens opacification) and other ocular degeneration are shown to be related to the [Ah] complex.
Toxification and detoxication of drugs and other environmental pollutants in extrahepatic tissues therefore can be important (1).
The eye is no exception. For example, chloroquine retinopathy observed in malaria patients was attributed to an accumulation of unusually high levels of chloroquine in the eye (2). Unless the eye possesses a mechanism for removing such drugs that enter ocular tissues via the circulating blood, therefore, accumulated chemicals of all types might exert adverse effects on photoreceptor cells, leading to visual impairment. Shortly after Bernstein's report (2), cytochrome P-450, a principal component of the drug-metabolizing enzyme system,* was detected spectrally in the pigmented epithelium of bovine retina (4). Although much progress has been made in studies on absorption, distribution, and clinical usefulness of ophthalmic drugs (5), information on ocular drug metabolism and toxicity remains quite scanty, in spite of obvious clinical importance.
Futher, information about genetic differences in ocular drug toxicity is even more scarce. Steroidinduced glaucoma is a pharmacogenetic disorder, for example, although the etiology is unknown. About 5% of the United States population is homozygous for the recessive allele causing glaucoma induced by corticosteroid ophthalmic medications (6).

P-450-Mediated Monooxygenase Activities and Other Coordinated Drug-Metabolizing Enzymes
Many environmental pollutants and other foreign compounds are chemicals that are so hydrophobic they would remain in the body indefinitely were it not for the metabolism resulting in more polar derivatives. These drug-metabolizing enzyme systems, which are localized principally in the liver, are usually divided into two groups: phase I and phase II. During phase I metabolism, one or more polar groups (such as hydroxyl) are introduced into the hydrophobic parent molecule, thus allowing a handle, or position, for the phase II conjugating enzymes (such as UDP glucuronosyltransferase) to attack. The conjugated products are sufficiently *In this paper, cytochrome P-450 is defined as all forms of CO-binding hemoproteins associated with membrane-bound NADPH-dependent monooxygenase activities. We define cytochrome P1-450 as all forms of CO-binding hemoprotein that increase in amount concomitantly with rises in induced AHH activity following polycyclic aromatic inducer treatment. In view of more than one such form of P1450 (3), it is emphasized that this definition of P1-450 is simplistic. 108 polar, so that these detoxified chemicals are now excreted from the body (7).
One of the most interesting of the phase I enzyme systems is a group of enzymes known collectively as the P-450-mediated monooxygenases (3). These membrane-bound enzyme systems metabolize polycyclic aromatic hydrocarbons such as benzo[a]pyrene (ubiquitous in city smog, cigarette smoke and characoal-cooked foods) and biphenyl; halogenated hydrocarbons such as polychlorinated and polybrominated biphenyls, insecticides, and ingredients in soaps and deodorants; strong mutagens such as N-methyl-N'-nitro-N-acetylarylamines and nitrofurans; numerous aromatic amines, such as those found in hair dyes; nitro aromatics and heterocyclics; wood terpenes; epoxides; carbamates; alkyl halides; safrole derivatives; certain fungal toxins and antibiotics; many of the chemotherapeutic agents used to treat human cancer; most drugs; small chemicals such as benzene, thiocyanate, or ethanol; both endogenous and synthetic steroids; and other endogenous compounds such as biogenic amines, indoles, thyroxine, and fatty acids.
Evidence is growing that metabolism to reactive intermediates by cytochrome P-450-mediated monooxygenases is a prerequisite for mutagenesis, carcinogenesis, and toxicity caused by numerous drugs, polycyclic hydrocarbons, and other environmental pollutants. The steady-state levels of these reactive electrophilic intermediates and, consequently, the rates at which they interact with the critical nucleophilic target, are dependent upon a delicate balance between their generation and detoxication ( Fig. 1). Changes in the balance between toxification and detoxication in any particular tissue of an individual may therefore affect his risk of tumorigenesis or toxicity.

Subcellular Localization and Distribution of Drug-Metabolizing Enzymes in the Eye
The distribution (Table 1) and subcellular localization (Table 2) of several drug-metabolizing enzyme activities were studied in bovine eye. AHH activityt was chosen as an example of phase I metabolism (reflecting basal forms of P-450). UDP glucuronosyltransferase, glutathione transferase, and Nacetyltransferase activities are typical phase II enzymes (9). y-Glutamyltranspeptidase and cystine tAbbreviations used include: AHH, aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1); B6, the C57BL/6N inbred mouse strain; D2, the DBA/2N inbred mouse strain. Ciliary body 5.74 396 3850 aTissue homogenates were used for assay of enzymic activities. One unit of activity is the amount of enzyme protein that produces 1 pmole of product per minute. Enzyme activities given here are specific activities, i.e., units/mg of tissue protein/minute. aminopeptidase are important in the pathway from glutathione conjugates of aromatic hydrocarbons to mercapturate formation (10). Most, if not all, of these six enzymes (Tables 1 and 2) are induced by various drugs or other environmental pollutants (3,9,10). Low levels of -y-glutamyltranspeptidase (11) and glutathione transferase (12) have been reported in the lens. We conclude from Tables 1 and 2 that virtually all ocular tissues have detectable drug-metabolizing capability (13)(14)(15). The ciliary body and pigmented epithelium-choroid are by far the richest in these activities (see Fig. 2 for the location of these tissues in the eye). In fact, the specific activities of AHH and UDP glucuronosyltransferase in the ciliary body are only about 10 to 20 times less than those in bovine liver.  the mouse regulates the induction (by polycyclic aromatic compounds such as 3-methylcholanthrene, benzo[a]pyrene, or 2, 3, 7, 8-tetrachlorodibenzo-pdioxin) of numerous drug-metabolizing enzyme "activities" associated with several (or many) new induced forms of cytochrome P1-450. The [Ah] complex comprises numerous regulatory, structural, and probably temporal genes that may or may not be on the same chromosome.
At any given dose of inducer, the induction of AHH activity and at least two dozen other monooxygenase activities and associated forms of P1-450 occurs in 3-methylcholanthrene-treated B6 and other genetically "responsive" inbred strains and is always much lower in 3-methylcholanthrene-treated D2 and other genetically "nonresponsive" strains. Besides the liver, this genetic expression is seen in such tissues as lung, kidney, intestine, lymph nodes, skin, bone marrow, pigmented epithelium of the retina (Table 3), brain, mammary gland, uterus, ovary and testis. The genetic response is therefore called "systemic," or occurring throughout virtually all tissues of the animal. Responsiveness to aromatic hydrocarbons has been designated the [Ah] complex: Ah is the dqminpnt allele; Ah is the recessive allele; the Ah IAh bhetQrozygote is phenotypically similar to the Ah lAh mouse in terms of degree of responsiveness. The trait of Ahresponsiveness among crosses involving B6 and D2 mice is therefore autosomal dominant (16).
Several studies indicate that the fundamental genetic difference resides in the Ah regulatory gene (Fig. 3), which encodes the cytosolic receptor capable of binding to inducers such as 3-methylcholanthrene, benzo[a]yprene, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. To our knowledge, only certain foreign chemicals bind to this receptor with saturability and high affinity (less than 1 nmole).
The B6 receptor appears to have at least 50 times better ?ffinity toward inducers of P1-450 than the receptor complex into the nucleus has been demonstrated in the Ah-responsive heterozygote and homozygote (18) and requires a temperaturedependent step (19). Discrepancies between the dextran-charcoal adsorption assay and the sucrose density gradient assay have been recently understood via chromatographic studies of the Ah receptor (20). What happens in the nucleus is not yet known (Fig. 3), but somehow the "information" (that these inducers. of P1-450 exist in the cell's microenvironment) is received; the response is transcription of specific mRNA's, translation of these mRNA's into specific enzymes such as P1-450, and incorporation of P1-450 into cellular membranes. These induced enzymes may aid in detoxication or they may April 1982 generate increased amounts of reactive intermediates.

The Ah Complex in Human Populations
In spite of shortcomings with the AHH assay in human cultured lymphoblasts (8,21), a growing list of clinical disorders appears to be associated with the human [Ah] complex. There clearly exists sufficient evidence that heritable variation of AHH inducibility occurs in man. Experimental difficulties, however, make it impossible at this time to be certain whether AHH induction is controlled by one or more genetic loci.   (24). Doses of more than 1000 mg of acetaminophen/kg body weight were almost always fatal to AhblAhd mice within the first 8 hr after acetaminophen administration, but these doses were not lethal, nor did they ever cause lens opacification to AhdlAhd mice. At lower doses of acetaminophen (400 to 800 mg/kg), the ocular opacity developed more slowly in Ahb/Ahd mice. If a cataract did not appear within 10 hr after acetaminophen administration, however, no cataract developed subsequently. The degree of opacification that had developed within these 10 hr was never reversible. (Reproduced with permission).

Correlation between the Ahb Allele and Lens Opacification Caused by Intraperitoneal Acetaminophen
Acetaminophen, a widely used analgesic-antipyretic agent, is metabolized to a toxic intermediate largely by some form of polycyclic aromatic-induced P1-450 (16). Therefore, we theorized that Ah-responsive 112 mice-following treatment with polycyclic aromatic inducers to enhance P1-450 levels-would display more acetaminophen toxicity than Ah-nonresponsive mice. This hypothesis turns out to be true not only in liver, where hepatic necrosis and covalent binding of acetaminophen metabolites occur (22), but also in the lens, where cataract formation was found (Fig.  4). An absolute correlation is foubnd (Fig. 5) between lens opacification and the Ah allele among four Ah-responsive inbred mouse strains, five Ah-Environmental Health Perspectives  (25). Each symbol represents an individual mouse. All mice had received 3-methylcholanthrene intraperitoneally 48 hr prior to intraperitoneal acetaminophen. The eyes were evaluated with an ophthalmic slit lamp 5 hr following acetaminophen treatment: a = no signs of opacification; 1 + = about 50% opacification; 2 + = complete opacification. The mice were then immediately killed, and the liver microsomal hydroxylase activity was determined.
nonresponsive inbred strains and among children of the B6D2F1 x D2 backeross. Figure 6 illustrates the effects of acetaminophen on glutathione concentrations in the liver and lens of3-methylcholanthrene-pretreated B6 and D2 mice. As expected, hepatic glutathione depletion is more pronounced in B6 than in D2 mice. Quite unexpectedly, however, lenticular glutathione levels are not depleted in either B6 or D2 mice following the large intraperitoneal dose of acetaminophen.
One possibility is that acetaminophen and its metabolites do not reach the lens and therefore glutathione is not depleted. This possibility was ruled out with the use of radiolabeled acetaminophen (Fig. 7). Similar kinetics of covalent binding occurs in both liver and lens protein, and in each case B6 tissues exhibit two to five times greater covalent binding than the corresponding D2 tissues.
How can covalent binding of acetaminophen metabolites occur in the apparent absence of any glutathione depletion? At least two possible answers come to mind. First, assuming glutathione depletion in the lens is necessary for cataract formation, one might envision some sort of compartmentalization of glutathione. In other words, although total lens glutathione concentrations are similar in B6 and D2 mice, a subcellular depletion of glutathione (directly April 1982 in the anterior subcapsular region of the lens where the cataracts develop) might occur in B6 but not in D2. Such a localized depletion might be augmented by a sudden influx of reactive intermediates (generated by the liver, ciliary body or whatever) so that local concentrations of the toxic agent far exceed local concentrations of glutathione in and near the anterior chamber. Alternatively, ocular toxicity may develop by a mechanism independent of glutathione levels. Electrophilic reactive intermediates could cause changes in cellular macromolecules in many ways: alterations in oxidations and reductions of endogenous molecules, free radical formation, permeability changes in the lens membrane, alterations in metabolism of endogenous substrates, etc. Very large doses of acetaminophen were used for the experiments illustrated in Figures 4-7. The only clinical situation that such large doses might be achieved would be attempted suicide. Relevant observations, however, have been recently reported. Upon hearing a seminar about the data in this report, Cohen and Burk (26) examined ten patients showing evidence of acetaminophen-induced hepatotoxicity due to self-inflicted overdoses. The age range was 17 to 45 years old, with only two women more than 23 years of age. One of these patients, a 45-year-old alcoholic about whom no previous medical history was available and who died 1 day later, was found on admission to have bilateral diffuse cataracts; no previous history of cataracts was known. She also was the only fatality in the series of 10 cases (26). 113 .  (15) 16.7 (15) 19.4 (15) 0 No cataracts aInjections twice weekly (25). 1'The drug was dissolved in corn oil and the food was soaked in this solution of corn oil. cEstimated drug concentration in corn oil in which the food was soaked. dMean body weights of group during feeding of the drug. The number of survivors is shown in parentheses.

Effect of Daily Oral Acetaminophen or Naphthalene on Cataract Formation
In additional experiments, we chose oral doses of acetaminophen, lower doses that are used clinically (Table 4). Oral naphthalene was also chosen for study because its monooxygenation is also catalyzed b!y some form of P1-450 and associated with the Ah allele (16). Naphthalene-induced cataracts have been described (27) in workers from dye and chemical factories. The cataractogenic agent is claimed (28) not to be naphthalene itself but rather 1,2-naphthoquinone, which is formed from naphthalene-1,2-dihydrodiol. This diol of naphthalene reaches the eye via the bloodstream. Pathways of 114 naphthalene metabolism have been extensively investigated both in vivo (29) and with liver microsomes in vivo (30)(31)(32). The phenol and the reactive arene oxide of naphthalene and acetaminophen are conjugated with glucuronide, sulfate, and glutathione.
Relatively small numbers of B6 mice develop detectable cataracts when acetaminophen or naphthalene is ingested daily (Table 4). Phenobarbital induces these conjugating pathways, and this fact may explain our finding (25) that oral phenobarbital can completely prevent acetaminophen-induced cataract formation. It is possible that the reactive intermediate of acetaminophen is an arene oxide or a quinone. No D2 mouse ever developed acetaminophenor naphthalene-induced cataracts. All mice treated with 3-methylcholanthrene died within 6 weeks, apparently due to the cytotoxicity of the Environmental Health Perspectives chemical when injected twice weekly. On the other hand, ,-naphthoflavone treatment was not very toxic; relatively few animals died during 60 days of daily oral acetaminophen or naphthalene combined with intraperitoneal P-naphtoflavone twice weekly.
Out of the nine B6 mice that developed cataracts on these various oral regimens, three remained otherwise healthy for 6 or 7 months, at which time the animals were killed in order to examine histologically the lenticular opacities.

Histology of Ocular Toxicity Induced by Oral Acetaminophen
Upon close microscopic examination, we found that tissue degeneration occurs not only in the anterior lens cortex but also in large areas of the uveal tract (Fig. 8a, c). The retina and pigmented epithelium are normal (Fig. 8b). The degeneration seen in the choroid, ciliary body and iris appears to be of a chronic nature. Anterior synechiae and inflammatory cells at the angle are frequently observed. The number of epithelial cells is usually decreased in cataracts that were young (several hours or several days old). Intermittent loss of cells from the lens epithelial layer is seen in cataracts that had been present for at least 1 week (Fig. 8c). April 1982 The gradual loss of the epithelial cells might result in metabolic derangements in the lens fibers, thereby leading to lens opacification. All lenses from D2 mice similarly treated do not show any evidence of ocular abnormalities.
Possible Interrelationship among Genetic Differences in Drug Metabolism, Drug-induced Cataracts and Senile Cataracts in the Human Population Cataracts induced by chemicals and drugs, especially naphthalene, have been extensively investigated because of their similarity to senile cataracts (28,33,34). The problem of trying to assess ocular toxicity in elderly patients receiving large daily doses of acetaminophen would be far more difficult than to study patients attempting suicide with acetaminophen, because ofthe combination ofgenetic factors (i.e., the [Ah] complex in man) and environmental factors (i.e., history of cigarette smoking, other drugs prescribed, dietary intake, etc.  Environmental Health Perspectives genetically determined small proportion of the total human population. We do suggest, however, that the ocular toxicity data shown in this report may be clinically important to certain patients receiving either a single large overdose of acetaminophen or high doses over a long period of time.