Effects of Heredity on Response to Drugs and Environmental

DECEMBER 1996 VOLUME 9, NUMBER 8 © Copyright 1996 by the American Chemical Society

Invited Review Effects of Heredity on Response to Drugs and Environmental Chemicals: Construction of Rodent Models Gerald N. Levy,* Lourdes Rodgers, and Wendell W. Weber Department of Pharmacology, University of Michigan, Ann Arbor, Michigan 48109 Received May 13, 1996

Why Do We Still Need Animal Models? With the emergence of molecular techniques in biology and in genetics in particular, the need for animal models in studies of pharmacology and toxicology is sometimes questioned. While it is possible (although often difficult) to do molecular analysis on genetically heterogeneous material, the use of genetically defined samples simplifies the procedures and the interpretation of the results. Isolating a molecule or sequencing a gene is only the beginning of a biological experiment; one must learn the function and regulation of the sequence in cell, tissue, and whole body (1). For example, we can clone the gene for an enzyme and transfect it into a cell. The enzyme catalyzes activation of a test compound, and perhaps, if the compound is a carcinogen, covalent binding to DNA occurs. This tells us nothing about the target tissue for the carcinogen and nothing about immune surveillance which may eliminate the mutated cell before tumor formation ever occurs. In an intact animal, however, all the physiological interactions between cells and tissues are present and the true determination of toxicity or carcinogenicity can be made. One chooses an animal model of the human response to a drug or carcinogen with the idea that it will contribute to the dissection of the genetic basis of the * Corresponding author: Gerald N. Levy, Ph.D., Department of Pharmacology, 1301 MSRB III, University of Michigan, Ann Arbor, MI 48109-0632. Phone: 313 763-2406; FAX: 313 763-4450; Email: [email protected].

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human response, to identify the biochemical and pharmacological mechanism responsible for the response, and to help in assessing the biological significance of the response. Animal models that turn out to be poor models for assessing a new drug therapy may still be excellent for elucidating the molecular and pharmacological basis of pathways involved in human disease. The process of modeling in animals involves going back and forth between the model and humans, comparing results between the two systems whenever possible. In humans, exposure to xenobiotics in general and to carcinogens in particular is difficult to measure. While therapeutic pharmaceuticals are a possible exception, most environmental xenobiotics are encountered at individually variable dosages. Genetic factors, age, sex, and other operant factors can greatly affect the pharmacokinetics and pharmacodynamics of an encountered compound. The influence of these factors on human response to a drug or carcinogen can often be examined by pedigree analysis, gene isolation, and structural analysis of the pharmacological or toxicological response. In animals, however, these limited methods can be supplemented by strain surveys, test crosses, studies in recombinant and congenic strains, and gene targeting. Studies in animals which are impossible in humans can reveal which cell types are involved in metabolism of the compound of interest, what are the key enzymes of the metabolic pathway, what genes are responsible for synthesis of the relevant enzymes, how these genes and their products are controlled, and whether there are © 1996 American Chemical Society

1216 Chem. Res. Toxicol., Vol. 9, No. 8, 1996

genetic differences that determine or modulate individual response to the compound. If there are differences in response between exposed individuals, and there usually are, studies under carefully controlled conditions in responders of differing known phenotypes and genotypes can determine the pharmacological and toxicological consequences of the response. By seeking correlations between the differences in response and differences in biochemical and molecular characteristics of the responders, important new insights into the effect of an exogenous chemical may be found. Ultimately, though, the extent to which findings in the animal model are applicable to humans must be evaluated.

Use of the Mouse as a Model for Human Response The mouse has been intensively studied for many years by genetic (1) and more recently by molecular genetic techniques. Genetic mapping of mice has moved ahead rapidly and is accelerating in the “molecular age”. Many biochemical as well as other polymorphisms have been mapped to specific locations on mouse chromosomes, and many areas of homology between mouse and human chromosomes have been found (2). Recombinant inbred, congenic, double congenic, and recombinant congenic mouse strains have become or are rapidly becoming standard tools for the analysis of individual response to environmental substances. Transgenic and “knockout” mutants created by gene targeting techniques developed even more recently have been applied to the analysis of a number of traits of pharmacologic and toxicologic interest, as will be tabulated below. Many inbred strains of mice have been established and extensively analyzed, and are available. Established colonies of these mice provide the opportunity to study large numbers of genetically identical individuals (if inbred mice are used) under strictly controlled environments and at lower cost than for most other laboratory mammals. Inbred strains of other rodents, particularly rats, provide an alternative to mice as models for human response in toxicology and carcinogenicity studies.

Spontaneous Mutations For many years investigators have had to be satisfied with animal models resulting from spontaneous mutations in populations of domesticated or laboratory species of animals. Genetic polymorphisms of the AH-receptor in mice and rats (3), cytochrome P450 variants in CYP2D6 and CYP2B1 homologues in rats (4, 5), the N-acetylation polymorphism in rabbits, mice, hamsters, and rats (6), the thiopurine methyltransferase polymorphism in mice (7), and sensitivity and resistance responses to bleomycin in mice (8) are a few examples of naturally occurring mutations. While these and other observations have led to useful models of human response, a number of problems are associated with naturally occurring models. Not infrequently the mutation is rare, making it difficult to find the appropiate model, the specific genetic defect may be difficult to identify and compare with its human counterpart, or the mutation occurs only in a species that is expensive or difficult to maintain. Nonetheless, these spontaneous mutations have often been the basis for construction of more useful inbred and congenic strains used to study the consequences of the mutated genotype.

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Use of Inbred Strains Some of the problems with models expressing spontaneous mutations can be alleviated through proper choice of species and a systematic comparison of genetic, biochemical, pharmacological, and toxicological characteristics of species and strains of laboratory animals to find traits which mimic those in humans. Inbred strains are rich sources for the development of animal models of human responses because of the long term stability of strain characteristics, genetic authenticity, phenotype uniformity, and the unique combination of genetic material possessed by individual strains. Continuous inbreeding by mating closely related individuals results in a gradual increase in genetic homogeneity until all individuals are genetically identical (1). A strain produced by 20 or more generations of full-sib matings is defined as an inbred strain. Many of the common inbred mouse strains have gone far beyond the 20 generations of brother-sister mating called for in this definition. Histories and characteristics of many inbred strains are available in the literature (9-11). Most of the desirable features of inbred animals are a consequence of the homogeneity and isogenicity that is achieved at nearly every gene locus through the prolonged inbreeding. As a result, inbred strains can provide an unlimited supply of replicate genotypes and can be manipulated to yield new combinations of genes that do not occur in nature. Many inbred strains of mice, hamsters, rats, guinea pigs, and even pigeons are available commercially or from research laboratories. Use of an inbred strain in studies of drug metabolism or carcinogenesis will produce data that are, for many traits, less spread about their mean value than when outbred animals are used because genetic differences between individuals are eliminated. On the other hand, it is often argued that, except for a few isolated populations, humans are outbred animals, and the great genetic diversity of the human population should be reflected in an animal model. Interpretation of experimental results from outbred animals is difficult. For example, the role of an enzyme being studied can be obscured by differences in other characteristics of the animal such as body mass, lifespan, gender, aging, as well as eating, drinking, and activity behavior. To study the response of an enzyme or receptor to a chemical, all other factors which might influence the response should be eliminated or reduced as much as possible. Confounding influences due to genetic factors are nearly eliminated in inbred strains, and environmental differences are eliminated by proper animal husbandry practices.

Strain Surveys for Spontaneous Mutations Not infrequently, a particular inbred strain shows a quantitatively or even qualitatively different response to a test compound than another inbred strain. Often certain inbred strains may have high activity of a particular enzyme, while other inbred strains will have low activity enabling strains to be classified into high or low activity groups. Examples of such strain surveys in mice include Ah receptor polymorphism (12), N-acetyltransferase activity (13), coumarin hydroxylase activity (14), and epoxide hydrolase activity (15). Offspring of crosses of a high activity and low activity strain may or may not have intermediate activity. However, all individuals of the F1 generation are heterozygous with 50%

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Figure 1. Production of recombinant inbred strains. Consider two inbred strains A and B, which differ at three unlinked loci E, F, and G.

of their genes inherited from the high activity strain and 50% from the low activity strain. Crosses within the F1 generation produce the F2 generation. All unlinked genes will be randomly distributed in the F2 individuals, and 20 generations of inbreeding of the F2’s will produce recombinant inbred strains. The concept of recombinant inbred strains is illustrated in Figure 1 and the following section. A more detailed discussion of recombinant inbred strains can be found in Weber (16).

Recombinant Inbred Strains Recombinant inbred (RI) strains are a set of strains that have been derived from the cross of two unrelated but highly inbred strains. If the two inbred strains are crossed, the F1 generation will receive half its genes from each strain making it heterozygous at each loci. The F2 generation will have a random distribution of all unlinked genes but a nonrandom distribution of linked genes. The tighter the linkage of genes, the less random will be their distribution in the F2 generation. Individuals from the F2 generation are chosen and inbred by fullsib mating for 20 generations to produce a set of recombinant inbred strains. Each recombinant inbred strain represents a stable population with all loci homozygous. The unlinked genes are distributed randomly across the strains, but closely linked genes are expected to be fixed in the same combinations as in the progenitor inbred strains (Figure 1). Notice that all genes are homozygous within each RI strain after 20 generations of strict brother-sister inbreeding. The value of recombinant inbred strains is to permit analysis of linkage between traits and, with appropriate markers, determination of chromosomal location for loci of interest. Genotype analysis of RI strains determines the strain distribution pattern (SDP) which enables the recombination frequencies of a gene of interest and various marker genes to be determined. The lower the frequency of recombination, the closer together on the chromosome are the two loci. For example, in their study of coumarin hydroxylase activity in mice, Wood and Taylor (14) genotyped a set of 24 RI strains produced from DBA/2J and C57BL/6J inbred strains for coumarin hydroxylase (Coh) and two other loci, glucose phosphate isomerase (Gpi-1) and hemoglobin β chain (Hbb), which differ in the inbred parental strains. By finding that there were 10 recombinations of Coh and Hbb in the 24 strains but only 4 of 24 recombinations of Coh and Gpi1, it was determined that Coh is closer to Gpi-1 than to

Figure 2. Order of genes on mouse chromosome 7 determined by SDP of 24 DBA/2J × C57BL/6J RI strains. The numbers indicate the recombination frequency (percent) observed between indicated loci.

Hbb. However, these experiments could not tell if the order of the loci was Coh-Gpi-1-Hbb or Gpi-1-Coh-Hbb. Use of an additional marker, the pink eye dilution marker (p), showed that there were significantly more recombinations between Gpi-1 and p (15/69) than between Coh and Gpi-1 (4/69) suggesting the distance between p and Gpi-1 is greater than the distance between Coh and Gpi-1. It was also known that p is distal to Gpi1, so the correct gene order is Coh-Gpi-1-p and the gene order of the 4 loci is Coh-Gpi-1-p-Hbb on chromosome 7 (Figure 2). Similar experimental approaches were used to map murine microsomal epoxide hydolase to chromosome 1 (15) and N-acetyltransferase to chromosome 8 (17).

Congenic Inbred Strains Comparisons between inbred strains which differ in a key enzyme of metabolism of a compound are useful in determining the role of that enzyme in toxicity or carcinogenicity of the compound. However, a given enzyme may be associated with an increase or a decrease in the response. Thus, low activity of epoxide hydrolase may increase or decrease toxicity from epoxides, low CYP1A activity may increase or decrease toxicity of polycyclic aromatic hydrocarbons, or low N-acetyltransferase activity may increase or decrease toxicity due to arylamines. The direction of change in the toxicity of a compound caused by alterations in enzyme activity is often tissue, sex, and species specific, and the effect may be subject to the influence of the activity of other enzymes of the metabolic pathway that act on a given compound. Inbred strains which differ in the activity of an enzyme of particular interest probably will also differ in many other enzymes as well as other hereditary factors that may also affect the response to the chemical. Genetic factors other than the one enzyme being studied are referred to as the genetic background. Ideally, comparisons should be made between individuals who differ only in the gene that codes the enzyme of specific interest. Congenic inbred strains enable us to approach this ideal.


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all (or almost all) of the genes identical except for the locus of interest.

Examples of Research Using Congenic Strains

Figure 3. Development of an acetylator congenic line. The B6 rapid acetylator line (rr) was crossed with the A slow acetylator line (ss). Heterozygous offspring (rs) were backcrossed to the A strain. The procedure was carried out for 12 generations, then the heterozygotes were crossed, and homozygous rapid acetylators were chosen to begin the congenic inbred A.B6Nat-r line. At each of the 12 backcrosses, half of the remaining B6 background genes are lost, so that the congenic strain is greater than 99.79% isogenic with A.

Congenic inbred strains are produced by crossing two inbred strains which differ at the locus of interest. Using the example of acetylator congenic strains (17), C57BL/ 6J (B6), a rapid acetylator, and A/J (A), a slow acetylator, were used to produce the A.B6Nat-r congenic strain (Figure 3). A, the background strain, was crossed with B6, the donor strain, to produce heterozygous F1 mice. In the backcross method used in this example, F1 mice were backcrossed with A mice and the individuals heterozygous for Nat were selected and backcrossed again with A. Backcrossing of heterozygous mice obtained at each generation was continued for 12 generations. At each generation the genes originally derived from the B6 strain are reduced by 1/2, except for the Nat gene which is identified (as the heterozygous phenotype) and preserved. Thus at the F1 generation, B6 genes ) 1/21 ) 50% and at the 12th generation, N12, B6 genes ) 1/212 ) 0.024%. At this point, N12, Nat heterozygotes are brothersister mated and the homozygous rapid acetylator offspring are selected for breeding, to found the congenic rapid acetylator line A.B6Nat-r. It can be shown that after the 12 generations of backcrossing, the length of chromosome containing the Nat locus that was transferred from B6 to A is about 15-20 centiMorgans. The “mirror image” slow acetylator congenic inbred strain, B6.ANat-s (B6.A), was obtained by a procedure similar to that just described. While a given inbred strain comprises individuals, all of whom are genetically identical, a pair consisting of an inbred strain and its congenic will have

Congenic inbred strains were originally designed by Snell (18) as a powerful tool to identify immunological factors related to the transplantation of tumors. Following the early success of congenic strains in the evaluation of single gene differences, their use has increased beyond immunology to include research in carcinogenesis and toxicology, teratogenesis, sensory and behavior phenomena, and growth and aging. A partial list of the areas of research in which congenic strains of mice, rats, and hamsters have been used recently is given in Table 1. The creation of acetylator congenic mouse strains was reported by Mattano et al. (17), and a complete verification of the transfer of the Nat gene from donor to background has been reported by de Leoń et al. (19). Acetylator congenic strains of hamster were developed by Hein et al. (20). The interest in acetylator congenic rodents arises from the applicability of the rodent model to study the human acetylation polymorphism and its role in toxicity and carcinogenesis (21). The association of rapid or slow acetylation with susceptibility to certain human diseases and toxicities has been known for several years (see monograph by Weber, (22). Although there are differences between the mouse and human polymorphisms, the similarities in the effects of the polymorphism enable use of the congenic mouse model to examine aspects not available for study in humans such as the relation of carcinogen dose to DNA adduct formation and persistence (23), tissue specificity of DNA adduct formation (24, 25), and the effect of gender and age at time of exposure on DNA-carcinogen adduct formation (26). Since the Nat gene is not the only determinant in the differential response of rapid acetylator B6 and slow acetylator A mouse strains to arylamines, the use of the congenic line B6.A was supplemented by its “mirror image” congenic line A.B6Nat-r and by the two inbred parental strains B6 and A. The four strains constitute a “quartet”. As shown in Figure 4, comparisons can be made to determine the effects of either the A or B6 genetic background on response to the differential allele. For example, comparison of strains A and B6 will show differences due to the background genes as well as the gene of interest (Nat in this example), whereas comparison of A with A.B6 or B6 with B6.A will show the effect of the Nat gene expressed on the A or B6 background, respectively. Comparison of A with B6.A or B6 with A.B6 will show the effects of the different backgrounds on slow or rapid acetylation, respectively. The value of such analysis is shown in Table 2. Mononuclear leukocytes isolated from mice were incubated with 2-aminofluorene and DNA-AF adducts were determined (27). Had only the inbred parental lines B6 and A been studied, no significant effect of acetylation differences would have been found. In contrast, comparing A with A.B6 shows a statistically significant greater adduct production in the rapid acetylator mice, and a similar result is observed in comparing B6 with B6.A. Additionally, the effect of background genes can be examined by comparing A with B6.A and B6 with A.B6. It is evident that the A background modifies the effect of Nat by increasing adduct formation compared to the same Nat allele expressed on the B6 background. While

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Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1219 Table 1. Areas of Research Using Congenic Rodent Strains

research field

species

trait studied

ref

cancer and toxicology

mouse mouse mouse mouse mouse mouse mouse mouse mouse hamster hamster rat rat mouse mouse mouse mouse mouse mouse mouse mouse mouse hamster

metabolism of heterocyclic amines porphyria from hexachlorophene 3-MC induced tumors N-acetylation N-acetylation N-acetylation N-acetylation mammary tumor viruses TNF release N-acetylation N-acetylation albumin and bladder cancer acetaminophen toxicity cleft lip ( palate drug induced cleft lip taste smell apolipoprotein and aging growth alcoholism acetylation analysis of congenics acetylation

Nerurkar et al., 1995 (24) Hahn et al. (42) Paigen et al. (43) Levy and Weber, 1990 (28) Levy and Weber, 1989 (44) Flammang et al. (45) Hein et al., 1988 (46) Matsuzawa et al. (47) Clark et al. (48) Feng et al. (49) Hein et al., 1992 (20) Takahashi et al. (50) deMorais et al. (51) Juriloff et al. (52) Karolyi et al. (53) Boughter and Whitney (54) Monahan et al. (55) Higuchi et al. (56) Lutz et al. (57) Dudek and Underwood (58) Mattano et al. (17) de Leoń et al. (19) Hein, 1991 (59)

Teratogenesis sensory phenomena growth and aging complex behavior producing congenics

Figure 4. A quartet of mouse strains consisting of two parental strains (A and B6) and two congenic strains derived from them (A.B6 and B6.A). Table 2. DNA-AF Adduct Formation (pmol of Adduct/mg of DNA) after 18 h Incubation of Mononuclear Leukocytes from Rapid and Slow Acetylator Mice with 60 µM AFa mouse strain

acetylator genotype

adducts formed

B6 B6.A A A.B6

RR (homozygous rapid) rr (homozygous slow) rr RR

2.38 ( 0.18 0.84 ( 0.10b 2.03 ( 0.16 3.45 ( 0.27c

a Data from ref 27. b Differs from B6, p < 0.01. c Differs from A, p < 0.01.

the quartet analysis can demonstrate the effect of background genes, it does not give information about which specific genes or mechanisms are responsible for the observed effects (other metabolic enzymes, repair mechanisms, etc.).

Double Congenic Strains More than a single enzyme is usually involved in metabolism of most chemicals encountered by an organism, and it follows that genetically polymorphic differences in activity of one of the enzymes of a given pathway

can be greatly modified by genetically polymorphic differences in other enzyme activities of the pathway. The contributions of two genetic polymorphisms to the toxicity of a compound can be investigated by an extension of the approach used for a single enzyme polymorphism through construction of double congenic inbred strains. The simplest way to make a double congenic strain is to cross two congenic strains having the same background. This can be illustrated by following the creation of the double congenic strain B6.A.D from a cross of B6.A and B6.D single congenic strains (28). B6.D is an inbred congenic strain having the allele for the AH receptor (Ahrb-1) of B6 mice replaced by Ahrd from the DBA inbred strain. B6 is responsive to TCDD, β-naphthoflavone (βNF), and similar P4501A inducers, but DBA is not responsive to these planar polycyclic aromatic hydrocarbons. Since the background of both strains is the B6 genome, individuals of the F1 generation of the B6.D × B6.A cross are homozygous for all loci except Ahr and Nat, for which they are heterozygous. F2 offspring of the F1 generation can have any of 16 combinations of Ahr and Nat alleles, assuming these traits are not linked. (As it turns out, the loci are not linked as Ahr is on chromosome 12 and Nat is on chromosome 8.) Phenotyping for nonresponders and slow acetylators among F2 offspring was expedited by the fact that both characteristics are recessive and thus only animals that are homozygous for the nonresponsive Ahr and slow acetylation alleles exhibit the characteristic. The expected frequency of the double homozygote is 1 in 16, so quite a few candidates must be produced and phenotyped as both male and female double homozygotes are required to found the double congenic strain. The construction of this double congenic strain is illustrated in the “checkerboard” diagram, Figure 5. In accordance with the standard vocabulary for double congenic strains, the double congenic created is designated B6.ANat-s.DAhr-d, or B6.A.D for short. The quartet of strains consisting of the double congenic, the parental, and the two single congenic strains can be used to determine the effects of one genetic region on another. Thus, the B6.A.D mice were used in conjunction with the parental B6 and single congenic B6.A and B6.D strains to investigate the influence of genetic variability


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Figure 5. Checkerboard diagram illustrating construction of the double congenic strain B6.A.D. Gene symbols are r: rapid acetylator, s: slow acetylator, b: Ah responsive, d: Ah nonresponsive.

generations, and inbreeding pairs of the offspring from backcross 2 or 3 to establish homozygosity in the resultant strains (29). The initial cross between inbred strains, call them A and B, produces offspring with all loci being a/b, that is 50% of genes are derived from B. Backcross 1 which is (A × B)F1 × A results in 25% of genes being derived from B, and each successive backcross to A reduces the percentage of B genes by 1/2 (12.5% after the second backcross and 6.25% if a third backcross is used). The probability of chance overlap in sets of transferred genes limits the amount of genome that can be covered with a set of RCS. With 12.5% of the genome transferred, 93% of the genome can be covered with a set of about 20 recombinant congenic strains. For a study of 4 relevant unlinked genes, the probability of more than one relevant gene being in the same strain is only 0.08 (29). The advantage of RCS is that unlinked components of a multigenic system are separated into individual inbred strains. Thus a multigene trait that depends on differential alleles at multiple loci is transformed into a set of single gene differences between the background strain and individual recombinant congenic strains. Individual RCS carrying different genes affecting the same trait can be crossed for studies of gene-gene interactions, and they can be used in combination with transgenic mice to determine the effect of the rest of the genome on the expression and function of the transgene. The chief limitation of RCS and congenic strains for such analyses is that only genes which differ in the parental strains can be analyzed. Recombinant congenic strains have been used to separate and analyze genes that affect the number and size of colon tumors produced by exposure to the carcinogen 1,2-dimethylhydrazine (30). They have also been used to separate multiple genes controlling T-cell response to IL-2 and anti-CD3 (31). Twenty RCS, produced from BALB/c and STS inbred strains, were used in these analyses, details of which have been published (32).

Figure 6. A quartet of mouse strains consisting of the background strain (B6), two “single” congenic strains (B6.D and B6.A), and a “double” congenic strain (B6.A.D).

Random Mutagenesis

at the Nat and Ahr loci on the formation of DNA adducts in liver after exposure to 2-aminofluorene (28), in colon, bladder, and kidney (24), and in liver and lung after exposure to IQ (25) with and without βNF treatment. As illustrated in Figure 6, comparison of pairs horizontally will demonstrate effects of genetic differences in the AH receptor (chromosome 12) on either rapid (upper row) or slow acetylators (lower row). Comparison of pairs vertically will demonstrate differences due to acetylation capacity (chromosome 8) in either responsive (left column) or nonresponsive mice (right column). The combined effects of deficits in both traits are demonstrated by the double congenic strain. Again, the other genes (the background) of these mice are identical since they are all derived from B6 inbred mice.

Exposing mice or other laboratory animals to mutagenic chemicals, irradiation, or retroviral insertion can greatly increase the rate of mutation above the normal spontaneous rate. The mutations produced are random, and careful analysis of expressed proteins or DNA must be performed (usually by electrophoretic techniques) to detect and identify the mutations. Only a small fraction of these random mutations will be of interest to the investigator, and a fair number of mutational events will be lethal. Consequently, random mutagenesis is more commonly used to determine the types of mutations caused by specific mutagenic agents rather than as a technique to create a given gene change. However, the high rate of mutations caused by certain mutagens (e.g., ethylnitrosourea) allows this technique to be used to screen for specific mutations.

Recombinant Congenic Inbred Strains

Gene Targeting

A combination of the recombinant inbred and congenic inbred approaches affords another way of investigating the individuality of genes involved in multifactorial traits. Recombinant congenic inbred strains (RCS) are produced by crossing two inbred strains, backcrossing the F1 generation to one of the parental strains for only 2 or 3

To truly understand the function of a gene, one must mutate it and compare the resulting organism with the normal organism. The most dramatic change that can be made in gene function is the complete elimination of function. Mutant mice which have been created by gene targeting so far usually contain null mutations of a given

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Chem. Res. Toxicol., Vol. 9, No. 8, 1996 1221 Table 3. Mouse Models Created by Gene Targeting

trait or defect

target gene

ref

spatial learning Duchenne muscular dystrophy aggressive behavior skeletal development pheochromocytoma Parkinson-like syndrome (not established) heart defects blood pressure regulation defective B6 metabolism lymphoid tumors epileptic seizures cystic fibrosis tyrosinemia (type 1) ulcerative collitis and colon cancer benzo[a]pyrene teratogenesis hemophilia A multiple tumor susceptibility chronic granulomatous disease neuronal degeneration gastric ulceration renal disease inflammation development

R-Ca-calmodulin kinase II D71 product of dystrophy serotonin (5-HT) receptor type X collagen neurofibromatosis (Nf1) D2 receptor R2C-adrenoceptor gap junction (connexin) eNOS alkaline phosphatase post-replication repair epilepsy (jerkey locus) Cftr (∆F508 locus) fumarylacetoacetohydrolase GRi2 subunit p53 factor VIII p53 superoxide production Alzheimer’s Aβ peptide PHS-1 PHS-2 PHS-2 AH receptor

Silva et al., 1992 (60) Greenberg et al., 1994 (61) Saudou et al., 1994 (62) Rosati et al., 1994 (63) Jacks et al., 1994 (64) Balk et al., 1995 (65) Link et al., 1995 (66) Reaume et al., 1995 (67) Huang et al., 1995 (68) Waymire et al., 1995 (69) Reitmair et al., 1995 (70) Toth et al., 1995 (71) Colledge et al., 1995 (72) Grompe et al., 1995 (73) Rudolph et al., 1995 (74) Nicol et al., 1995 (75) Bi et al., 1995 (76) Harvey et al., 1995 (77) Pollack et al., 1995 (78) LaFerla et al., 1995 (79) Langenbach et al., 1995 (80) Morham et al., 1995 (81) Dinchuk et al., 1995 (82) Fernandez-Salguero et al., 1995 (83)

gene. These models afford a promising way to study mammalian traits, but a number of questions require control over the timing of gene expression as well as expression in specific tissues. The creation of mice which possess less drastic mutations, or mutations targeted to specific organs or cell types, would be even more informative. New strategies for inducing tissue-specific expression (33) such as using the adipocyte P2 promoter for selective expression in fat cells, the myosin light-chain promoter for expression in muscle, the amylase promoter for expression in the acinar pancreas, or the insulin promoter for expression in islet β cells (34) and for temporal manipulation of gene expression by a tetracycline-responsive promoter (35) are proving useful in answering such questions. To be successful, gene targeting needs to be directed, affecting only the locus to be studied. Thus targeting needs to be specific such that controlled changes are made which will alter the control, stability, or functional activity of the gene product. In addition, the process requires some minimum efficiency to ensure practical significance of the procedure (36). Careful design of the vectors to be inserted into the mouse zygote is neccessary (33, 34, 36). In addition to changes in the DNA of the gene of interest, genes (and their promoters) for selectable markers must be designed into the vector for transfection. These markers allow the transfected cells that contain the mutated gene to be identified and enriched.

Transgenic Mice Although zygote injection can be used in the production of transgenic mice, embryonic stem cells (ES) are currently preferred for targeting experiments. The advantages of ES cells include that they can be screened for homologous recombination events, homologous recombination occurs at high frequency in these cells, and they can be returned to a “host” embryo and be incorporated into the developing mouse where the genetic information of the ES cells can be distributed to cells of all tissues including germ cells. A majority of studies using transgenic mice have involved replacing a normal gene with a null allele (inactive) thus creating a “knockout” mouse. By deter-

mining what functions or characteristics of an organism are altered by lack of a known gene, one can learn what are the normal functions of the missing gene. Table 3 presents examples of the recent papers on investigations of gene function through gene targeting.

Analysis of Multigenic Traits by Quantitative Trait Loci Mapping Many of the genetic characteristics of importance in studies of drug and carcinogen metabolism are not “all or nothing” responses but rather quantitative modifiers of response (e.g., susceptibility factors). While basic Mendelian theory of inheritance explains discrete, qualitative differences between individuals, observations show that most traits exhibit continuous variation. Such quantitative differences can result from the segregation of multiple monogenic factors, modified by environmental factors (37). Quantitative trait loci (QTL) mapping has emerged as a useful method to determine the number and nature of genes involved in such multifactorial traits in mice and rats, the only species for which sufficiently dense genetic linkage maps have thus far been developed. The basic procedure for mapping QTLs is to cross two inbred strains having a large difference in some quantitative trait followed by scoring the segregating progeny (either from an F1 × Parent backcross or from an F1 × F1 intercross) for both the trait and for a number of genetic markers (37). Phenotype and genetic marker information can then be used for “interval mapping”. The term “interval mapping” is short for the estimation of the probable genotypes and most likely QTL effect at every point in the genome from phenotypic and genetic marker information (38). Each such map is scored for its probability (“likelihood”); the best map is the one with maximum likelihood. Currently, QTL mapping is being more widely used to determine the chromosome location of genes, to estimate the number of loci associated with a complex trait, to identify important modifier genes, and to estimate the percentage of phenotypic differences due to genetic versus environmental factors. Examples of the use of QTL mapping include analysis of airway hyperresponsiveness


in mice (39), determination of a modifier gene in intestinal hyperplasia in mice (40), and mapping of a gene causing hypertension in spontaneously hypertensive rats (41).

Summary Metabolism of xenobiotics including drugs and carcinogens is an important determinant of the effect of these chemicals on the responses of the organism. A complex interplay of activation and detoxification reactions, of conjugation reactions, of sequestering and elimination, of progression of toxicity or repair of damage all determine the ultimate effect of xenobiotic exposure. The enzymes responsible for each step of these processes are produced from information encoded in the gene for the enzyme, and thus, the expression and effect of the enzyme can be studied by genetic techniques. Animal models, and particularly rodent models have been and continue to be extremely useful in defining and understanding the biological significance of human response to chemicals. From studies of rare, spontaneous mutations in metabolizing enzymes to the more sophisticated techniques using inbred, recombinant inbred, congenic inbred, and recombinant congenic inbred strains, rodent models have provided a powerful analytical approach to otherwise unobtainable information on the role of enzymes in the disposition and metabolic activation of drugs and carcinogens. The newer techniques of gene targeting, use of transgenic mice, and QTL mapping in mice and rats have greatly expanded the experimental approaches available to investigate the role of the metabolic enzymes, receptors, and other variant proteins of interest. It is even possible through inducible gene targeting to determine the role of a gene product at different stages of development or in specific tissues (33, 35). Research using techniques outlined in this review will continue to expand as new targets for drug action are proposed and discovered.

Acknowledgment. Work in the authors’ laboratories has been supported by NIH Grants CA39018, GM44965, OH00081, and 2T32 GM07544.

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