Genetic mechanisms for variability in drug response and toxicity.

It is now well established that many proteins involved in the metabolism or pharmacodynamic action of drugs and foreign compounds exhibit structural polymorphism and variation in their level of expression. This variation leads to dramatic phenotypic differences in response to medicines or susceptibility to carcinogenesis. Some of the changes in the phenotypic expression of proteins are secondary to variation in the nucleic acid sequence of their respective genes. The science of pharmacogenetics links differences in gene structure (polymorphism) with pharmacologic differences in drug action and disposition of foreign compounds. Through discussion of four examples, we will emphasize the variety of genetic mechanisms that can potentially influence the phenotypic response to xenobiotic challenge and pharmacotherapy. The first example illustrates how structural variation in the coding region of drug metabolizing enzymes influences risk of drug toxicity. A second example demonstrates how genetic variation can influence gene transcriptional regulation and how the resulting dysregulation may be linked to increased susceptibility to exposure-linked cancer. The third example illustrates how genetic polymorphism can selectively influence the pharmacodynamic response to medication, and the final example of warfarin response illustrates how genetic variation in more than one gene can account for broad extremes in phenotypic response.

Genetic mechanisms for hypersensitivity and resistance to the anticoagulant Warfarin.

Warfarin is the therapeutic of choice for maintenance anticoagualtion therapy. A principle caveat of this medication is that the dosage required to achieve the desired therapeutic effect varies up to 120-fold between individuals. Currently, there are no reliable means of prospectively identifying which patients will require either unusually high or low dosages. This dilemma puts patients at risk of therapeutic failure or potentially life-threatening overdosage during a prolonged trial-and-error period of establishing an individualized medication strategy. Pharmacogenetic research has revealed that extreme differences in the drug dose required to achieve the desired therapeutic response can be attributed to genetic variation in the genes encoding drug metabolizing enzymes, and cellular receptor proteins. The anticoagulant Warfarin represents a model system where there is evidence to suggest that both pharmacokinetic and pharmacodynamic mechanisms contribute to the overall variability in patient response. Here the current understanding concerning the influence of genetic variation in Warfarin pharmacokinetics is reviewed and the potential for similar genetic mechanism impacting on the pharmacodynamic response in man is explored. Diagnostic testing to identify subjects requiring low-dose Warfarin therapy is discussed in light of potential confounding or coexisting resistance to the drug effects.

Detection of cocaine metabolite in serum and urine: frequency and correlation with medical diagnosis.

Review of toxicology screening results in our level 1 trauma center revealed that approximately 15% of urine drug screens were positive for cocaine metabolite. Our objective was to determine the prevalence of true acute cocaine intoxication and if measurement of serum would improve upon the accuracy of toxicology screening for identifying acute cases of cocaine poisoning. Cases were analyzed for cocaine metabolite (benzoylecgonine, BE) in matched serum and urine specimens and for acute cocaine intoxication by retrospective chart review. BE was identified in 3.8% of serum and 14.6% of urine specimens. For the identification of the acutely poisoned patient, the predictive value of toxicology screening was 53.4% for serum and 17.8% for urine. Of patients who screen positive for BE in serum, the odds of intoxication are 44:1 (95% CI 4.85-396.7). We conclude that subjects who test positive for BE in serum have a high probability of acute cocaine intoxication and that analyzing serum improves the predictive value of testing by 3-fold.

Fundamentals and applications of pharmacogenetics for the clinical laboratory.

Metabolism and disposition of foreign compounds, including pharmaceuticals, is dependent upon a host of factors. However, a genetic basis for individuality in drug metabolism has long been recognized and more recently has been confirmed. It is now well understood that a finite number of inherited sequence variants (alleles) of genes encoding drug-metabolizing enzymes give rise to discrete drug metabolism phenotypes. This primer in pharmacogenetics will introduce the clinical laboratorian to the mechanistic basis underlying the influence of genetics on pharmacology. We begin with an overview of pharmacology and introduce the importance of protein structure in maintaining steady-state drug concentrations. After review of fundamental concepts related to drug-metabolizing enzymes and genetics, we then give examples of how discrete genetic variations (polymorphism) alter the response (phenotype) to certain therapeutics in select individuals. We conclude with several analytical and interpretive considerations which must be considered by laboratories offering pharmacogenetic services.

Pharmacogenetics in the practice of laboratory medicine.

BACKGROUND: The clinical laboratory forms an essential bridge between fundamental discoveries in biological sciences and their transition into effective medical practice. The genetic basis for individuality in drug metabolism and response is the result of a finite number of inherited sequence variants (alleles) of genes encoding drug-metabolizing enzymes and drug receptors. Pharmacogenetics (PG) links differences in gene structure with pharmacological differences in pharmacokinetics and pharmacodynamics. The next step in the process of applying PG (or pharmacogenomic) information to individualized therapeutic management is dissemination of this information to practicing physicians by clinical laboratorians. Transitioning PG analysis into clinical practice will require professionals in laboratory medicine to identify relevant polymorphisms, develop sensitive and specific testing strategies, and, in conjunction with physicians and pharmacologists, communicate interpretive guidelines regarding appropriate indications for testing and rational dose adjustment. We review these concepts and provide examples of how PG can be applied to support therapeutic decision making.

Negative regulation of the rat glutathione S-transferase A2 gene by glucocorticoids involves a canonical glucocorticoid consensus sequence.

Glucocorticoids (GCs) repress both basal and polyaromatic hydrocarbon-induced expression of the glutathione S-transferase Ya1 gene (gstA2) in isolated rat hepatocytes and rat liver in vivo. Transient transfection experiments with HepG2 cells were used to identify GC-responsive elements (GREs). With cotransfected GC receptor, chloramphenicol acetyltransferase (CAT) constructs containing a palindromic GRE (pGRE) and three GRE hexanucleotide half-sites between -1.6 and -1.1 kb of the 5'-flanking region of gstA2 were repressed >50% by GC when induced with polyaromatic hydrocarbon. This pGRE, if either mutated or deleted, significantly reduces GC responsiveness of the gene to 20-30%; no effect of GC was observed with CAT constructs containing -1.15 kb of the 5'-flanking region. The dexamethasone concentration dependence of the repression was consistent with involvement of the GC receptor and was antagonized by RU38486. Electrophoretic mobility shift assays demonstrated that pGRE formed a specific DNA/protein complex, which was prevented by the addition of excess unlabeled or mouse mammary tumor virus GRE but not by unrelated or mutated gstA2 GRE double-stranded oligonucleotides. This complex was supershifted by incubation of nuclear extracts containing GC receptor with anti-GC receptor globulins. Constructs containing multiple copies of pGRE sequence were either nonresponsive or positively responsive (three copies) to GC. Luciferase constructs containing -1.62 to -1.03 kb of the 5'-flanking region also were regulated positively by GC. Chimeric GC-peroxisome proliferator activated receptor activated the constructs that were positively responsive to GC but did not mediate the negative effect in constructs containing 1.6 kb of 5'-flanking region. We conclude that pGRE and half-site GREs of gstA2 participate in regulation of this gene; however, a second unidentified responsive element must exist between -1.03 and -0.164 kb, resulting in repression of gstA2 expression.

Standards of laboratory practice: antidepressant drug monitoring. National Academy of Clinical Biochemistry.

Therapeutic drug monitoring (TDM) for certain tricyclic antidepressants (TCAs) and lithium is supported on the basis of clearly defined therapeutic ranges. TDM is of particular importance in individuals whose pharmacokinetic behavior may differ from that of the general population or is changing as the result of aging and maturation. Once steady-state drug concentrations are achieved, serum or plasma specimens should be collected during the terminal drug-elimination phase and separated from cellular blood components immediately. Methods of analysis must be specific for parent drug and active metabolites and demonstrate imprecision (CVs) within 5-10% over the therapeutic range. For support of overdose situations, semiquantitative values for TCAs and quantitative measures of lithium should be available within 1 h, and routine TDM results should be reported within 24 h of receipt in the laboratory. Standardized and rigorous laboratory practices contribute to improved therapeutic management.

Pharmacogenetics: a laboratory tool for optimizing therapeutic efficiency.

Pharmacogenetics is the study of the linkage between an individual's genotype and that individual's ability to metabolize a foreign compound. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Phenotypes exhibiting poor and ultraextensive metabolism result from genetic variance (polymorphism) of enzymes involved in metabolism. Thus, in pharmacogenetic studies one applies genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug metabolism phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic efficiency. More than 25 commonly prescribed medicines are metabolized by the cytochrome P-4502D6 (CYP2D6) isoenzyme, and polymorphism of the CYP2D6 gene affects the therapeutic management of up to 17% of individuals in some ethnic groups. In this review, we summarize and update information concerning drug-metabolizing genotypes with emphasis on CYP2D6 genotyping techniques that can be applied by the clinical laboratory for linking human genetics to therapeutic management.

Hormonal regulation of hepatic enzymes involved in foreign compound metabolism.

The regulation of hepatic P450s has been the focus of numerous studies because of the importance of these proteins in endocrinology, oncology, and toxicology, as well as drug development. Considerable evidence exists demonstrating that many hepatic P450s are regulated by developmental, sex, or hormonal factors in addition to receptors that interact with foreign chemicals. The focus of work in our laboratory has been on the effects of steroid hormones, especially glucocorticoids, on expression of genes regulated by the Ah receptor. We have shown that most rat hepatic genes of the Ah receptor gene battery are regulated by glucocorticoids. We have used glucocorticoid-deficient animal models to demonstrate that these steroids do modulate the expression (basal and inducible) of these genes in vivo. Using cultured rat hepatocytes, we have demonstrated that polycyclic aromatic hydrocarbon (PAH) induction of cytochrome P4501A1, glutathione S-transferase Ya1, and UDP-glucuronosyltransferase 1*6 are apparently potentiated two- to fourfold upon inclusion of glucocorticoids in the media to activate the glucocorticoid receptor and further, that the receptor antagonist RU 38486 reverses these phenomenon. NAD(P)H:quinone oxidoreductase and aldehyde dehydrogenase 3 gene expression were repressed 70-80% by glucocorticoids in cultured hepatocytes through a glucocorticoid receptor-mediated process as well. The effect of glucocorticoid concentration on PAH induction of glutathione S-transferase Ya1 subunit for glucocorticoids was biphasic, but at physiological concentrations gene expression was repressed to approximately 20-40% of control. At supraphysiological concentrations, glucocorticoids alone induced expression two- to threefold and potentiated the PAH-inducible expression of the Ya1 subunit gene. Subsequent work in our laboratory has focused on defining the molecular basis of this hormonal regulation, specifically elucidating responsive elements responsible for the action of the glucocorticoid receptor and the mechanisms by which some of these genes are positively regulated and others are negatively regulated.

Mechanism and elimination of aspirin-induced interference in Emit II d.a.u. assays.

The presence of salicylates in urine reduces the signal in Emit assays (Syva), potentially yielding false-negative drugs-of-abuse screening results. We demonstrate that the principal urinary metabolite of salicylate, salicyluric acid (SUA; 2-hydroxybenzoylaminoacetic acid), interferes with the measurement of NADH formed in the assay by reducing the molar absorptivity of NADH at 340 nm. Thus, for a given concentration of d.a.u. analyte the change in absorbance over the assay time interval is less in the presence of SUA. With the Emit cocaine assay on the Hitachi 704 analyzer, the rate of absorbance change (delta AR) monitored at 340 nm for a specimen containing approximately 270 micrograms/L benzoylecgonine (BE) was 57 +/- 1.9 mA/min without SUA and 29 +/- 2.7 mA/min with 5 g/L SUA (n = 20). In contrast, delta AR determined at 376 nm was 18.6 +/- 0.5 mA/min with and 17.9 +/- 0.8 mA/min without 5 g/L SUA (n = 20). Measuring the Emit assay signal at wavelengths where SUA has no absorbance (376 nm) eliminates the interference due to SUA while maintaining the precision of the assay near the cutoff concentration for BE (300 micrograms/L).

Decreased signal in Emit assays of drugs of abuse in urine after ingestion of aspirin: potential for false-negative results.

During routine drug analysis with the Syva d.a.u. Emit immunoassays we observed a high frequency of urines with lower rates of changes in absorbance (delta A R) than the rate for a drug-free urine calibrator. Many of these urines contained salicylates. Among 40 urines with apparent salicylate concentrations between 15 and 420 mg/dL tested for benzoylecgonine (BE), 20 had delta A R < -4 (range +2 to -28 mA/min). The rates decreased with increasing salicylate: delta A R = -0.057 x (salicylate, mg/dL) -0.22 mA/min (r = 0.85, n = 40, P < 0.01). Urines from 100 control subjects (no salicylate) had mean +/- SD delta A R values of -1.05 +/- 2.2 mA/min (range +3 to -7; only two were < -4 mA/min). Although direct addition of salicylic acid (200 mg/dL) to urine specimens did not reproduce the negative bias, ingestion of aspirin (acetylsalicylic acid) did by -0.09 mA/min per 1 mg/dL (72.4 mumol/L) salicylate. Negative biases observed for other Emit d.a.u. assays after salicylate ingestion lead us to conclude that ingestion of therapeutic doses of aspirin may cause false-negative results for drug screens in urines by this technology.

Developmental aspects of glucocorticoid regulation of polycyclic aromatic hydrocarbon-inducible enzymes in rat liver.

The expression of hepatic cytochrome P4501A1 (P4501A1), glutathione S-transferase Ya subunit (GST), and NAD(P)H:quinone oxidoreductase (QOR) proteins was evaluated in fetal, neonatal, and adolescent rats treated with 3-methylcholanthrene (MC) and the synthetic glucocorticoid dexamethasone (Dex) to elucidate the developmental aspects of glucocorticoid regulation of the induction of drug metabolizing enzymes by polycyclic aromatic hydrocarbons in vivo. These developmental states were chosen to represent either glucocorticoid deplete or replete conditions due to their differences in circulating glucocorticoid levels. Rats were treated with either MC (10 mg/kg body wt) or Dex (10 mg/kg body wt) or a combination of both and sacrificed 24 h later. In neonatal rats, the enzyme activities of P4501A1, GST, and QOR were increased by MC treatment approximately 65-, 1.4-, and 7-fold, respectively. The induction of these enzymes by MC was further potentiated an additional 2-, 1.5-, and 1.4-fold by concomitant Dex treatment. In adolescent male rats, Dex potentiated MC induction of P4501A1 activity (1.7-fold), but repressed MC induction of GST and QOR activities. When the protein contents for the three enzymes were measured by Western blot analyses, a positive correlation was observed with enzyme activities for all conditions except for the adolescent rat, where hepatic protein content of P4501A1 of rats treated with both MC and Dex was not significantly increased above the level seen with 3-methylcholanthrene treatment alone. The levels of specific mRNA and transcriptional activity for cytochrome P4501A1, GST Ya isozyme, and QOR closely paralleled the changes seen in their protein content in the livers of neonatal and adolescent rats. Dexamethasone potentiation of P4501A1 expression at the protein and RNA level were clearly statistically significant in the neonatal rat, but not in the adolescent rat, suggesting that the circulating levels of glucocorticoids are sufficiently low during the neonatal period that the full expression of induction of P4501A1 was not attained in the absence of exogenously administered glucocorticoids. These data also demonstrate that glucocorticoids have differential effects on the induction of GST Ya subunit and QOR protein and RNA in the neonatal and adolescent state, possibly related to circulating levels of glucocorticoids.

Potentiation of 3-methylcholanthrene induction of rat hepatic cytochrome P450IA1 by dexamethasone in vivo.

The potentiation of the expression of polycyclic aromatic hydrocarbon-inducible cytochromes P450IA1 and P450IA2, and phenobarbital-inducible cytochrome P450IIB1 and NADPH-cytochrome P-450 reductase by dexamethasone in vivo was investigated using adrenalectomized and sham-operated rats. Hepatic cytochrome P450IA1 activity (determined by ethoxyresorufin O-deethylase activity) was induced maximally (30-fold) in both sham-operated and adrenalectomized rats 24 hr after a single injection of 3-methylcholanthrene (10 mg/kg). Dexamethasone (10 mg/kg) increased hepatic tyrosine aminotransferase activity 3- to 4-fold, but had little or no effect on ethoxyresorufin O-deethylase activity. However, dexamethasone potentiated the 3-methylcholanthrene-dependent induction of hepatic P450IA1 activity 2- to 3-fold in adrenalectomized rats and 1.5- to 2-fold in sham-operated rats when administered concomitantly with 3-methylcholanthrene (P less than .05). The dose of dexamethasone required to potentiate 3-methylcholanthrene induction of P450IA1 activity (greater than 1 mg/kg) correlated well with the dose required to induce hepatic tyrosine aminotransferase activity; a marker of glucocorticoid action. Potentiation of 3-methylcholanthrene induction of P450IA1 activity in the rat appears to be tissue specific in that dexamethasone was found not to potentiate 3-methylcholanthrene induction of P450IA1 activity of rat lung or kidney. Cytochrome P450IA1 content induced by 3-methylcholanthrene in the liver was also potentiated 2- to 3-fold by dexamethasone. In contrast, potentiation of 3-methylcholanthrene induction of cytochrome P450IA2 content was not observed. Western blot analyses indicate that the increase in monooxygenase activity induced by 3-methylcholanthrene or 3-methylcholanthrene plus dexamethasone corresponds well only with the induction of cytochrome P450IA1 protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

Bilateral rhegmatogenous retinal detachment associated with unilateral choroidal melanoma.

A patient with bilateral rhegmatogenous retinal detachment associated with a unilateral choroidal melanoma is presented. It appears that there is no causative relationship between these two findings in this case. The clinician should be advised that these two phenomena are not as rare as was once thought, and an index of suspicion should be maintained whenever one is evaluating a patient with retinal detachment.