23

Responses to Drugs

Hans Meier and John L. Fuller

Mice of randombred stocks are widely used in the pharmaceutical industry for the testing of drugs. This chapter, however, concentrates on genetic variation in responses to drugs as observed in inbred mice, hybrids, and mutant stocks. Differences of this kind may affect drug assays and, more importantly, shed light on the mechanism of drug action. Some mutant stocks may be useful for assessing drugs for their curative, ameliorative, or prophylactic effects. A survey of this one species illustrates the role of genotype in determining responses to drugs and also exemplifies some of the unsolved problems of pharmacogenetics.

GENETICS OF RESPONSES TO DRUGS

The existence of strain differences in response to drugs has been amply documented. The mode of transmission of response patterns has been less investigated and few of the differences observed have been related to a single locus with major effects. The quantitative genetics of pharmacological reactions is necessarily complex because of the multiplicity of possible phenotypes for study ( Green and Meier, 1965). One cannot simply assume that the intensity of all observed responses to a particular drug will be determined by the same hereditary makeup.

Most of the human pharmacogenetic literature ( Kalow, 1962) concerns single-locus effects on sensitivity related to sex-linked differences in glucose-6-phosphate dehydrogenase ( Price-Evans and Clarke, 1961). In contrast, studies of strain differences have predominated in the mouse, though a few single-locus systems have been reported. For example, Dagg et al. ( 1964) found single-locus control of the rate of metabolism of the pyrimidines, uracil, dihydrouracil, and thymine.

The analysis of the responses of inbred, hybrid, and randombred mice to insulin ( Brown, 1961a) and to sodium pentobarbitone ( Brown, 1961b) illustrates the quantitative approach to strain differences. A randombred strain, LAC, was generally less sensitive to insulin than were inbred strains. Hybrids between several of the inbred strains were compared with respect to ED₅₀ and mean slope of the dose-response regression line ( Table 23-1). It is apparent from the table that the outcomes of the two crosses, one of them made reciprocally, follow no simple rule. The hybrid may resemble one parent in ED₅₀ and neither in average slope. Similar results were obtained with pentobarbitone sodium ( Brown, 1961b). Brown states, "The ability to forecast the usefulness of any F₁ hybrid for a particular pharmacological assay will depend on prior knowledge of the response of its parents, but the confirmatory experimental results may be disappointing."

When sensitivity in assay can be correlated with other strain characteristics, the factors responsible for variation in pharmacological response may be indicated. There is a significant relationship between average body weight and ED₅₀ for insulin ( Figure 23-1A), but the correlation between weight and slope is less clear ( Figure 23-1B).

GENETICS AND BIOASSAYS

The choice of animals for bioassay procedures has been widely debated ( Becker, 1962). A part of the argument has centered upon the relative merits of inbred mice and their F₁ hybrids. There is some evidence that hybrids are phenotypically more uniform than inbreds ( Chapter 9). Thus, given equal sample size, one should expect greater reproducibility from hybrids. On the other hand, hybrids may possess better homeostatic mechanisms and thus be less affected by the administration of a drug or hormone ( Chai, 1960). It is not unlikely that the desiderata of uniformity and sensitivity may be negatively associated. Thus the choice of animals for bioassay is a matter for empirical determination. Likewise one has a choice of statistics for judging the results of a bioassay. Chai ( 1960) has used λ = s/b as a measure of efficiency, where s is the standard deviation within groups and b is the regression coefficient of the dose-response curve. He found no superiority of hybrids over inbreds in a number of hormone assays. Becker ( 1962) compared drug responses by an F-statistic, related to λ, based on analysis of variance.

Apparently, then, there is no general rule for selecting the most sensitive assay animal in advance. Randombred mice are more widely used than either inbreds or hybrids in commercial assay work, where not only the sensitivity but the cost of subjects is important. Careful cost studies might show that the greater uniformity of inbred and F₁ hybrid mice would more than compensate for the added expense of their production. Becker and Chai have both suggested that stocks of mice selected on the basis of specific drug sensitivity might be valuable for assay purposes. At least one such strain — a stock of Swiss mice with a high incidence of audiogenic seizures — has proved valuable in testing anticonvulsant and tranquilizing drugs ( Swinyard et al., 1963). The further development of such stocks is a matter of economics. It is almost certain that selection can produce strains with increased or decreased specific drug sensitivities as needed.

It is sometimes argued that heterogeneous mice are more desirable for toxicity testing, since the range of sensitivities provides a better chance of detecting idiosyncratic responses which may be serious. The merit of this point of view is questionable. The occurrence or nonoccurrence of exceptional toxic responses in heterogeneous mice is not conclusive with respect to idiosyncratic toxic responses in man. The advantages of genetic uniformity and the consequently increased reliability of toxicity measurement seem to be greater than the advantages of heterogeneity.

VARIATION IN PHARMACOLOGICAL REACTIONS

Variations in pharmacological response have been found using a wide variety of drugs and strains of mice. It seems likely that any broad survey of mouse strains will uncover significant differences. The following sections describe examples of differential responses grouped according to the pharmacological or chemical characteristics of the substances used. Additional cases of variation in response to chemical agents are found in Chapters 9, 32, and 33.

Central nervous system stimulants

The need and importance of specifying the strain of mice used in studies on drug effects is emphasized by Weaver and Kerley's ( 1962) investigations of the response of several strains to d-amphetamine. Confirming the reports of other investigators Weaver and Kerley showed that aggregated mice are more susceptible than isolated mice to the lethal effects of amphetamine.

Mice tested were the Swiss-Webster, C57BL/6, DBA/2 strains and B6D2F₁ hybrids. The median lethal dose (LD₅₀) of amphetamine was different in each. Aggregated Swiss-Webster mice were five- to 10-fold more susceptible than isolated mice, but no evidence for increased lethality was found in aggregated C57BL/6 mice. Piperacetazine (2-acetyl-10-(3-4-beta-hydroxyethyl piperidino) phenothiazine), phenobarbital, and metaglycodol (2-m-chlorophenyl-3-methyl-2,3-butanediol) reduced the lethal effects of amphetamine in aggregated Swiss-Webster mice and not in aggregated B6D2F₁ mice. Also, B6D2F₁ mice appeared more susceptible than Swiss-Webster mice to metaglycodol, piperacetazine, and the convulsant effects of pentylenetetrazol, but less susceptible to strychnine.

Insulin

Chase et al. ( 1948) first reported a high tolerance of mice to insulin; mice of the KL strain survive about 300 times the dose lethal for LT/Ch mice. Apparently the cause of the difference is the presence of an insulin-destroying enzyme "insulinase" in the livers of KL mice which protects them from extreme hypoglycemia ( Beyer, 1955). Quantitative differences in insulin tolerance are also found in other strains ( Brown, 1961a). Among nine strains tested C57BR/cd was by far the most sensitive (650 milliunits/kg); DBA/2, BALB/c, and A2G were alike in sensitivity (900 milliunits/kg). Sensitivity was measured by the approximate ED₅₀ convulsive responses.

Serum from obese mice ( ob/ ob) has greater insulin-like activity than serum from normal littermates, yet the glucose tolerance of obese mice is normal ( Mayer et al., 1953). In contrast, NZO (New Zealand Obese) mice reveal a typical diabetic type of glucose intolerance in about two-thirds of the individuals ( Crofford and Davis, 1963). The mice having normal glucose tolerance tests were consistently normal; those that showed a impaired glucose tolerance showed a consistent impairment. The average blood sugar concentration of NZO mice is 22 mg/100 ml higher than that in mice of the control strain. Insulin sensitivity tests revealed them to be four to five times more resistant to insulin-induced convulsions than the controls.

Hormones other than insulin

Clear evidence for a genetic basis of hormone response in mice has been presented by Chai ( 1960) and by others ( Chapter 20). The response of C57BL/6J, C57BR/cdJ, and their F₁ hybrids (B6BRF₁) to androgen (testosterone propionate) was measured by the weights of seminal vesicles in 4-week-old castrated males. There appeared to be no difference in the slope of the three dose-response curves. Results of chorionic gonadotropin assays in 1-month-old females of strains DBA/1J, DBA/2J, and A/J were as follows: the regression of the dose-response curve was largest for BALB/cJ and least in A/J; that of the CAF₁ hybrid fell between that of the parental strains, but close to the A. The difference in slope between the two DBA strains, though not large, seemed to show genetic divergence as a consequence of separation for more than 20 generations.

The thyroid provides an additional example of heritable variations in hormonal activity. Iodine metabolism differs between inbred strains of mice and their F₁ hybrids as determined by several physiological measures ( Chai, 1958; Amin et al., 1957).

In the course of the development of an assay for adrenal cortical hormones it was noted that adrenalectomized mice (maintained on salt solution) responded to epinephrine in two different ways, some showing a decrease in eosinophila and others an increase during a period of 3 to 4 hours following subcutaneous injection ( Speirs and Meyer, 1949). Those that responded with eosinophilia turned out to have accessory adrenals and could survive after removal of the extra salt. After one dose of epinephrine the eosinopenic response became refractory to ACTH, histamine, etc., but not to 11-oxy-corticosteroids. Thus Speirs ( 1953) used epinephrine pretreatment to prepare mice as assay subjects for cortisone. It is well known that accessory adrenal cortical tissue is present in many mammals. Although knowledge of presence or absence, incidence, etc. of accessory nodules should be required by those suing animals for experiments involving adrenalectomy, pertinent information is available only for inbred mice and certain of their hybrids ( Hummel, 1958; Chapter 13).

The finding that growth rate, protein turnover, and nitrogen requirement are heritable suggests that strains of mice differ in their ability to secrete pituitary growth hormone or in their sensitivity to this hormone ( Russell and Bloom, 1956). There is no direct chemical method for the determination of growth hormone, but cardiac glycogen is, within limits, an indicator of growth hormone activity ( Russell and Bloom, 1956). At room temperature the cardiac glycogen of food-deprived mice was lowered. Large doses of growth hormone were required to maintain a normal level in fasting mice. At 30°C the cardiac glycogen of A strain mice increases during a 24-hour fast and administration of growth hormone increases the glycogen level still further. At this elevated temperature fasting lowers the cardiac glycogen of the I strain mice, and growth hormone raises the glycogen above the level of fed animals. It was suggested that a fasting stimulus, requiring the mediation of the hypophysis, evokes a greater secretion of growth hormone in A strain mice ( Adrouny and Russell, 1956) or that the tissues of these mice are more sensitive to the hormone ( Fenton and Duguid, 1962).

The availability of hairless mice has recently given impetus to cosmetological hormone research. Two types of hairless mice, one suitable for study up to the age of about 4 months and the other suitable up to about 2 months of age were used by Homburger et al. ( 1961). Several materials successfully smoothed the skin by hydration and densification of dermal connective tissue. Among them were the estrogens, estrogen and progesterone, ethisterone, and prenenolone. Testosterone made the skin look rougher. Microscopically, the beneficial effects consisted of densification and hydration of the dermis with widening of its papillase and consequently stretching and flattening of the skin folds. Interestingly, a commercial cream base produced a desirable effect through hydration of epidermal cells. When hormonal ingredients were given in solvents rather than the cream base there was complete absence of epidermal swelling.

Clearly then, this study not only holds considerable promise for assay studies, but also has opened up avenues for more basic investigations in cosmetic research dealing with mechanisms of action rather than simply clinical effectiveness of compounds.

Psychotropic drugs

After treatment with iproniazid, high mortality and hepatic injury occurred in AKR mice, whereas at the same dosage there was no evidence of toxicity in C57BL/6 and DBA/2 mice ( Rosen, 1951). Mice carrying the dilute genes ( d, d^l), such as the P strain and the sublines of DBA (DBA/1 and DBA/2), are both phenylketonuric and subject to the same audiogenic seizures ( Chapter 19). The alleles d and d^l have a depressant effect upon phenylalanine hydroxylase activity; d causes about a 50 per cent and d^l about an 85 per cent inhibition ( Coleman, 1960). Meier ( 1963a, 1963b) reported large differences in iproniazid toxicity between mice of the following genotypes, +/+, d^l/+, and d^l/ d^l. For example, after treatment with iproniazid phosphate LD₅₀'s were 1010, 976, and 715 mg per kilogram of body weight, respectively, for the three genotypes. Iproniazid did not prevent convulsions, but enhanced them both in rapidity of onset and severity. In contrast dl-α-ethyl-tryptamine (Monase) prevented the seizures. The cause of the failure of iproniazid is still hypothetical but the possibility exists that it may elevate brain catecholamines rather than serotonin. It may be pertinent that medmain, an antiserotonin compound, has been shown to induce seizures in mice ( Woolley, 1959).

Protection against sound-induced convulsions (audiogenic seizures) was obtained in noninbred Swiss mice by various phenothiazine ataractics ( Plotnikoff and Green, 1957; Plotnikoff, 1958, 1960), whereas chlorpromazine was entirely inactive in an inbred strain of Swiss mice ( Plotnikoff, 1960). Changes in drug resistance were observed during the course of inbreeding ( Plotnikoff, 1961). Chlorpromazine exerted diminishing protection from parental to each succeeding generation; a similar trend was observed with other phenothiazines, promazine, perphenazine, prochlorperazine, and trifluoperazine. In contrast, sodium phenobarbital uniformly gave protection against convulsions in all inbred generations tested. The lowered response to protective effects of chlorpromazine in each succeeding generation was very probably the result of selection or to chance fixation of recessives during the inbreeding.

Considerable variation in sensitivity to chlorpromazine has been found among the widely used inbred strains. In one study subjects were adult mice of both sexes from C57BL/6J, DBA/2J, A/HeJ, C3HeB/FeJ, and all their possible hybrids ( Huff, 1962). Differential drug sensitivities were revealed by use of tests of activity after administration of chlorpromazine, 1 to 4 mg/kg intraperitoneally. With 4 mg/kg, 90 per cent of strain C57BL/6J mice, but only 7 per cent of strain C3HeB/FeJ were inactive in an open-field test. A/HeJ and DBA/2J mice showed complete depression in 62 per cent and 67 per cent of the individuals, respectively. Results from study of hybrids indicate that a simple genetic mechanism, possibly involving no more than two loci, could be responsible. Weight differences were not correlated with differential suppression of activity nor was the suppression of exploratory activity correlated with appearance of ataxia ( Huff, 1962).

A mutant with spastic symptoms (gene symbol spa) that lends itself for use in assessment of sedative drugs was discovered by Chai ( 1961). Chai et al. ( 1962) found that aminooxyacetic acid abolished the symptoms whereas dilution was only moderately effective. Hydroxylamine and trimethadione were totally ineffective.

Catecholamines

Few data are available concerning the normal organ content of adrenaline and noradrenaline in mice. Owing to fundamental differences in the techniques of measurement, comparison of the results of different studies is extremely difficult. However, De Schaepdryver and Preziosi's ( 1959) investigation of the effect of drugs on the adrenaline and noradreneline content of adrenal glands, heart, liver, and spleen of normal adult white inbred mice will be mentioned. In some extracts of heart and spleen no adrenaline could be detected, suggesting that it may not be a regular constituent of these organs in mice and that its occurrence may depend on the presence of chromaffin cell groups irregularly scattered throughout the organism. Resperpine, insulin, nicotine, and histamine (in order of decreasing activity) lower the catecholamine content of the mammalian adrenal gland. After depletion of adrenaline and noradrenaline the time required for restoration of the catecholamines may be remarkably long. The rate of resynthesis apparently depends on the substance used and not on the degree of depletion. Of the two reserpine-like drugs used in this study only the one with sedative action, desmethoxyreserpine, caused complete depletion of noradrenaline in the adrenal gland. The amount of this catecholamine was only transiently lowered after reserpiline, which is supposedly devoid of tranquilizing properties. Sedative does of chlorpromazine, mepazine, perphenazine, and promazine provoked only a slight depression of adrenal catecholamines, as did sedative doses of meprobamate. This implies that sedation can occur without depletion of adrenal noradrenaline. Furthermore, the data presented proof of the preferential depletion of adrenaline and noradrenaline after pharmacological stimulation. Iproniazid, which largely prevents catecholamine depletion after reserpine by blockade of monoamine oxidases, also protected against adrenal gland catechol depletion provoked by other pharmacological agents.

Lack or decreased amounts of catecholamines in adipose tissue from genetically obese mice may be responsible for one of their metabolic defects, decreased lipolysis, since mobilization of free fatty acids from adipose tissue is regulated in part by catecholamines and ACTH. However, although fat depots of normal mice have been measured, there are as yet no measurements for obese mice. Sidman et al. ( 1962) found that noradrenaline is present in the epididymal white fat and especially in the interscapular brown fat of the mouse. They reported values of 0.05 and 0.49 μg per gram of wet tissue, respectively. Adrenaline levels were only about 10 per cent of those obtained for noradrenaline.

Determination of adrenal epinephrine and norepinephrine levels in normal and dilute-lethal ( d^l/ d^l) mice disclosed differences with respect to both total amounts and a rate of increase. During the period from birth to 3 weeks of age there is a rapid rise of both amines, but at a greater rate and to higher levels in the mutant. By 21 days dilute-lethal mice have about 25 per cent more epinephrine and over twice as much norepinephrine as do normal mice ( Doolittle and Raunch 1965).

Antioxidants

Antioxidants have been found to prolong lifespan of mice. The half-survival time of C3H mice was prolonged by 2-mercapto-ethylamine hydrochloride (1 per cent by weight, incorporated into pellets) from 14.5 to 18.3 months, an increase of 26 per cent, while hydroxylamine hydrochloride (1 per cent by weight) produced a slight prolongation, 7 per cent. Cysteine hydrochloride (1 per cent by weight) and hydroxylamine hydrochloride (2 per cent by weight) increased the half-survival time of AKR male mice from 9.6 to 11.0 and 11.2 months, respectively, an average prolongation of about 15 per cent. Ascorbic acid (2.0 per cent) and 2-mercapto-ethanol (0.5 per cent) had no significant effect ( Harman, 1961).

None of the antioxidants studied, 2-mercaptoethylamine hydrochloride (1 per cent by weight), 2,2' diaminodiethyl disulfide (1 per cent by weight), and hydroxylamine hydrochloride (1 and 2 per cent by weight) produced a marked decrease in the tumor incidence of C3H female mice. This latter finding suggests the possibility of prophylactic cancer chemotherapy by this or other anticancer agents. Similarly, encouraging results with reducing agents against Ehrlich's ascites tumor have been reported. In addition to the compounds already mentioned, N-methyl-formamide and potassium arsenate were found to produce tumor inhibition. The use of antioxidants tested the hypothesis that endogenously produced free radicals such as HO· and HO₂·, contribute both to aging (Harman, 1956a, 1956b, 1957) and to the incidence of spontaneous tumors ( Harman, 1961), since reducing substances could act as free radical inhibitors.

Purines and pyrimidines

Investigations comparing the susceptibilities of various strains of inbred mice to teratogenic stimuli are numerous ( Chapter 14). For example, 12-day embryos of two strains, BALB/c and 129, were approximately equally sensitive to 5-fluorouracil (30 to 40 mg/kg). However, strain 129 was more sensitive at all ages to a dose of 20 mg/kg. At this dosage effective teratogenic concentrations of fluorouracil apparently do not reach the BALB/c embryos or, alternatively, these embryos readily inactivate the drug. If the blood concentration is the critical factor, then the resistance of the BALB/c mice may be due to a slow rate of uptake from the peritoneal fluid or to rapid rates of catabolism or excretion.

Purines or pyrimidines apparently influence lipolysis. Dole ( 1961) found that they (as well as caffeine and pyrophosphate) increased the lipolytic action of epinephrine, adrenocortical-stimulating hormone (ACTH), thyroid-stimulating hormone (TSH), and glucagon. Indeed obese mice treated with a combination of purines, caffeine, and epinephrine gained less weight than untreated controls (Meier, 1963a, 1963b).

Carcinogens

Urethan (ethyl carbamate) has been reported to augment the induction of lymphoid leukemias by X-rays, estrogen, and cholanthrene ( Doell and Carnes, 1962). Since urethan alone did not augment the incidence of tumors in low-leukemia strains, it may be classified as a co-leukemogen. The drug has been found not to affect the incidence of leukemia in the high-leukemia strains, AKR and C58. In Swiss albino mice injected at birth or given toxic doses in the drinking water as adults, urethan augmented the leukemia incidence and shortened the latent period. Later it was shown that administration of urethan to newborn C57BL/6 mice induced thymic lymphomas with a high frequency approached only by that following divided doses of whole-body irradiation. Urethan also induces pulmonary tumors in certain strains of mice, among them strain BALB/c. Reciprocal lung grafts from BALB/c and DNA (1 day old) to their F₁ hybrids treated with urethan revealed that susceptibility to the carcinogenic action of urethan is determined by the intrinsic properties of the lung graft rather than the host ( Shapiro and Kirschbaum, 1951). Young rapidly growing Swiss mice are more sensitive to the tumorigenic effects of urethan than mice just arriving at maturity. It has been suggested that urethan brings about the adenomatous state by acting upon the nucleus of alveolar lining cells ( Rogers, 1951).

Considerable progress has been made in the study of the inheritance of spontaneous and induced experimental tumors. It has long been known that the inheritance of susceptibility to lung tumors is controlled by many genes. Tumors appear when the combined effects of genetic and nongenetic factors surpass a physiological threshold (Heston, 1942a, 1942b). Chemical carcinogens are potent factors which greatly increase the probability of the occurrence of the tumors above that are characteristic of a particular strain when untreated. Agents studied included dibenz (a, h) anthracene. 3-methylcholanthrene, urethan, nitrogen, sulfur mustard ( Heston, 1950), and radiation ( Lorenz et al., 1946). High concentration of inhaled oxygen has been found to increase the number of pulmonary tumors in the susceptible strain A mice injected with dibenz (a, h) anthracene over that in mice injected likewise but kept in air (reviewed by Heston, 1956). Bloom and Falconer ( 1964) confirmed the multifactorial inheritance of susceptibility to lung-tumor development in mice. However, they clearly demonstrated that about three-fourths of the difference in susceptibility between the C57BL/Fa and A/Fa strains is attributable to a single gene, ptr (pulmonary tumor resistance). The presence of a single recessive major gene conferring low susceptibility was established in analysis of all possible F₁ hybrids between six inbred strains, A/Fa, C57BL/Fa, RIII/Fa, JU/Fa, and KL. Strain C57BL which had the lowest susceptibility behaved differently from the other strains in crosses; it di not give F₁'s with intermediate susceptibilities, but behaved as a "recessive" in all the crosses ( Chapter 9).

Miscellaneous chemicals

Jay ( 1955) determined the mean sleeping time produced by hexobarbital (125 mg/kg) in 12 inbred strains. Values ranged from 18 minutes in SWR/HeN to over 48 minutes for A/LN. Ambrus et al. ( 1955) found that Swiss ICR mice are 6.6 times more sensitive to the effects of histamine than C3H/J. Strain and sex differences in response to serotonin (5-HT) were found for a number of strains. For example, a dose of 135 mg/kg of 5-HT creatine sulfate intraperitoneally killed half of the animals in a group of C3H/HeJ in 24 minutes, whereas 98.5 mg/kg killed half of the C57BL/6J in 34 minutes; these animals had been adrenalectomized prior to the treatments (Meier, 1963a, 1963b). Exposure of mice of several strains to minute amounts of chloroform in the air results in potentially fatal kidney lesions in males but less so in females. Death may follow exposure by as little as 1 hour, but there may be a delay of several weeks. In such cases the lesions are of the same type as those in early death. Greatest susceptibility to chloroform is found in strains C3H, C3Hf, A, HR, and DBA. Male mice of C57BL/6, C57BR/cd, C57L, and ST are resistant to amounts of chloroform that are lethal to the mice of the strains listed above ( Deringer et al., 1953; Shubik and Ritchie, 1953).

SUMMARY

Responses to drugs are in part influenced by hereditary factors. In some of the examples of hereditary determination of reactions to drugs, the differences between strains are probably due to many pairs of genes; others are apparently due to single pairs of genes. The over 200 inbred strains of mice with the large number of known genes and the many mutant stocks provide suitable materials to study the genetic basis for differential drug responses.

¹The writing of this chapter was supported in part by Public Health Service Research Grants CA 04691 from the National Cancer Institute and MH 01775 from the National Institute of Mental Health.

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