Significant alterations of biodistribution and immune responses in Balb/c mice administered with adenovirus targeted to CD40(+) cells.

CD40 ligation has been shown to promote antigen-presenting functions of dendritic cells, which express CD40 receptor. Here we reported significantly altered biodistribution and immune responses with the use of CD40-targeted adenovirus. Compared with unmodified adenovirus 5, the CD40-targeted adenovirus following intravenous administration (i.v.) resulted in increased transgene expressions in the lung and thymus, which normally do not take up significant amounts of adenovirus. Intradermal injection saw modified adenovirus being mainly processed in local draining lymph nodes and skin. Following intranasal administration (i.n.), neither unmodified nor targeted viruses were found to be in the liver or spleen, which predominantly took up the virus following i.v. administration. However, inadvertent infection of the brain was found with unmodified adenoviruses, with the second highest gene expression among 14 tissues examined. Importantly, such undesirable effects were largely ablated with the use of targeted vector. Moreover, the targeted adenovirus elicited more sustained antigen-specific cellular immune responses (up to 17-fold) at later time points (30 days post boosting), but also significantly hampered humoral responses irrespective of administration routes. Additional data suggest the skewed immune responses induced by the targeted adenoviruses were not due to the identity of the transgene but more likely a combination of overall transgene load and CD40 stimulation.

Cytochrome P4502C9 (CYP2C9) allele frequencies in Canadian Native Indian and Inuit populations.

CYP2C9 is the major P450 2C enzyme in human liver and contributes to the metabolism of a number of clinically important substrate drugs. This polymorphically expressed enzyme has been studied in Caucasian, Asian, and to some extent in African American populations, but little is known about the genetic variation in Native American populations. We therefore determined the 2C9*2 (Arg144Cys) and 2C9*3 (Ile359Leu) allele frequencies in 153 Native Canadian Indian (CNI) and 151 Inuit subjects by PCR-RFLP techniques. We also present genotyping data for two reference populations, 325 Caucasian (white North American) and 102 Chinese subjects. Genotyping analysis did not reveal any 2C9*4 alleles in the CNI, Inuit, Caucasian, or Chinese individuals. The 2C9*2 allele appears to be absent in Chinese and Inuit populations, but was present in CNI and Caucasian subjects at frequencies of 0.03 and 0.08-0.15, respectively. The 2C9*3 allele was not detected in the Inuit group, but occured in the CNI group (f = 0.06) at a frequency comparable to that of other ethnic groups. This group of Inuit individuals are the first population in which no 2C9*2 or *3 alleles have been detected so far. Therefore, these alleles may be extremely rare or absent, and unless other novel polymorphisms exist in this Inuit group one would not anticipate any CYP2C9 poor metabolizer subjects among this population.

A reverse transcription-polymerase chain reaction bioassay for avian vitellogenin mRNA.

A bioassay based on the measurement of vitellogenin (VTG) mRNA in avian embryo hepatocyte cultures by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was developed. To allow sequence comparison and design of suitable PCR primers, a short region of VTG cDNA was cloned and sequenced for seven species of birds. Cell cultures were prepared from both chicken and herring gull embryos and treated with the estradiol analogue moxestrol or the organochlorine insecticide o,p'-DDT. Using primers based on an area of the VTG gene that was identical for herring gull and chicken, in vitro VTG mRNA induction was observed for both moxestrol- and o,p'-DDT-treated cultures. Herring gull embryo hepatocyte cultures responded with VTG mRNA induction at moxestrol concentrations of 1 nM compared with 10 nM in chicken embryo hepatocyte cultures. Both herring gull and chicken embryo hepatocyte cultures responded with substantial VTG mRNA induction when treated with 10,000 nM o,p'-DDT. These results suggest that the bioassay will be useful for comparing avian embryo hepatocyte culture concentration-response data in terms of intra- and interspecies sensitivities to pharmacological estrogens or environmental contaminants.

Evidence for the catalysis of dextromethorphan O-demethylation by a CYP2D6-like enzyme in pig liver.

The pig is increasingly being used in pharmacological and toxicological studies, and is the species of choice for future research into xenotransplantation, extracorporeal liver support and hepatocyte-based bioartificial liver. However, relatively little is known about xenobiotic-metabolizing enzymes in this species. In the present study, immunoblotting with polyclonal anti-rat and anti-human cytochrome P450 (CYP) antibodies revealed the presence of proteins in pig liver which cross-reacted with anti-human CYP1A2, CYP2D6 and CYP3A4, and with anti-rat CYP2E1 antibodies. Northern blot analysis demonstrated the presence of mRNA which hybridized to cDNA probes for human CYP2D6, CYP2E1 and CYP3A4, and to an oligonucleotide probe for pig CYP3A29. As there is a lack of a good animal model for CYP2D6, the presence of a CYP2D6-related protein in pig liver was of particular interest. Pig hepatocytes also demonstrated CYP2D6 immunoreactive protein, and mRNA hybridizable to a CYP2D6 cDNA probe. We investigated the ability of pig liver microsomes to catalyse dextromethorphan O-demethylation, a widely-used marker enzyme activity for CYP2D6. This enzyme activity demonstrated biphasic kinetics, with a high affinity apparent K(m1)=6.9+/-3.6 microM and V(max1)=10.5+/-6.1nmol/min/nmol CYP. The reaction was sensitive to inhibition by the CYP2D6-selective inhibitors quinidine, quinine, lobeline and norfluoxetine, whereas chemical inhibitors selective for other CYP isoforms failed to affect the reaction. We conclude that dextromethorphan O-demethylation is catalysed by a CYP2D enzyme which is remarkably similar to human CYP2D6, suggesting potential value of the pig as a model for predicting human metabolism of xenobiotics which undergo CYP2D6-dependent biotransformation.

Detection of quantitative trait loci affecting caffeine metabolism by interval mapping in a genome-wide scan of C3H/HeJ x APN F(2) mice.

Caffeine metabolite ratios have been widely used to measure cytochrome P-450 1A2 activity in humans. Serum paraxanthine/caffeine ratio is one such index of this activity. We had previously demonstrated genetic variation of this trait among inbred mouse strains. In the present study, we have undertaken a genome-wide scan for quantitative trait loci affecting this trait with an interval mapping approach on an F(2) intercross population of acetaminophen nonsusceptible and C3H/HeJ inbred mice. A statistically significant association (log-likelihood ratio = 25.0) between a locus on chromosome 9, which colocalized with the murine Cyp1a2 locus, and the plasma paraxanthine/caffeine ratio was identified. This result suggested the presence of an expression polymorphism affecting this gene. A second locus was identified on chromosome 1 (log-likelihood ratio = 9.7) for which no obvious candidate gene has been identified. The influence of this locus on the paraxanthine/caffeine index was more significant among males (log-likelihood ratio = 6.3) than females (log-likelihood ratio = 3.6). A third locus was identified on chromosome 4 with a less statistically robust association (log-likelihood ratio = 3.4) to the paraxanthine/caffeine phenotype. Collectively, these three loci accounted for 63.2% of the variation observed in the F(2) population for this phenotype. These results demonstrate the potential for genetic variation arising from factors other than CYP1A2 activity to influence the plasma paraxanthine/caffeine ratio in mice. This study demonstrates the utility of quantitative genetics in the analysis of polygenic drug metabolism.

Regulation of cytochrome P4501A metabolism by glutathione.

Gene expression of cytochrome P4501A (CYP1A) in the rainbow trout Oncorhynchus mykiss is dependent on aromatic hydrocarbon receptor signal transduction, and is markedly sensitive to tissue thiol status. Tissue glutathione (GSH) status was manipulated by exogenous GSH, L-buthionine-[S,R]-sulfoximine (BSO), lipoate or 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Tissue GSH contents were significantly elevated in GSH- and lipoate-supplemented trout. Hepatic, renal and plasma GSH levels were markedly arrested in BSO-treated trout. Oxidized glutathione (oxidized GSH) levels were significantly elevated in the BCNU-supplemented group. Both BCNU treatment and BSO-induced GSH deficiency increased steady-state levels of hepatic CYPIA mRNA. Additional exposure to 0.1 mg/kg 3,3',4,4'-tetrachlorobiphenyl marginally suppressed the tetrachlorobiphenyl-dependent CYP1A induction in BSO-treated livers compared with the respective thiol treatment groups. Tetrachlorobiphenyl exposures altered efficiencies of thiol treatments and increased oxidized GSH content in all but the BSO-treated groups. However, exposure to 5 mg/kg tetrachlorobiphenyl altered effects of thiol treatments on CYP1A mRNA to a small extent, but catalytic activity of CYP1A was many times suppressed in BSO-treated and lipoate-supplemented fish. These results suggest that thiol status interferes with CYPIA metabolism in a two-way mode of action and provide further evidence for a cross-talk between cytochrome P4501A and glutathione.

Increased basal expression of hepatic Cyp1a1 and Cyp1a2 genes in inbred mice selected for susceptibility to acetaminophen-induced hepatotoxicity.

Susceptibility to acetaminophen-induced hepatotoxicity was found to vary widely in an outbred colony of Swiss Webster mice. Some acetaminophen-treated male mice showed a significant elevation in serum levels of the hepatic enzyme alanine aminotransferase at a normally non-hepatotoxic oral dose. A selective breeding program over 17 generations produced inbred mice which were either susceptible or nonsusceptible to the hepatotoxic effects of acetaminophen. Liver microsomes from the susceptible group showed a statistically significant increase in the ability to metabolize acetaminophen to a reactive intermediate which covalently binds N-acetylcysteine. Microsomal cytochrome P450 activities associated with CYP1A2 (acetanilide 4-hydroxylation and methoxyresorufin O-demethylase) were significantly increased in the susceptible group. Ethoxyresorufin O-deethylase activity, associated with both CYP1A1 and CYP1A2, was also significantly elevated in this group. Further examination of both CYP1A isoforms revealed that hepatic CYP1A1 and CYP1A2 mRNA and protein levels were significantly elevated in animals from the susceptible group. In vivo caffeine 3-demethylation, which is associated with CYP1A2 activity, co-segregated with acetaminophen susceptibility and showed a significant positive correlation (r = 0.626, p < 0.005) with CYP1A2 mRNA expression in animals from both the susceptible and nonsusceptible groups. The co-segregation of elevated basal Cyp1a1 and CYP1a2 gene expression levels in animals selected for susceptibility to acetaminophen-induced hepatotoxicity suggested a common heritable basis for regulation of basal expression of both of these CYP1A isoforms. This was supported by the correlated expression of both CYP1A mRNAs within individual mice (r = 0.644, p < 0.02).

Regulation of 3,3',4,4'-tetrachlorobiphenyl induced cytochrome P450 metabolism by thiols in tissues of rainbow trout.

We observed that glutathione (GSH) status regulates the Ah receptor inducible cytochrome P4501A (CYP1A) gene expression and catalytic activity in 3,3',4,4'-tetrachlorobiphenyl (TCB) exposed rainbow trout. Tissue GSH status of TCB (1 mg/kg body weight, in corn oil) injected fish was manipulated by a) injecting (i.p.) GSH (0.25 g/kg), b) arresting GSH synthesis by L-buthionine-[S,R]-sulfoximine (BSO; 6 mmol/kg) injection for 3 and 6 days. Our attempt to manipulate GSH levels by lipoate supplementation (16 mg/kg) was not productive. Both BSO- and lipoate-supplemented fish maintained a low tissue redox (GSSG/GSH) ratio. Activities of glutathione peroxidase and glutathione reductase were elevated following 3 days of GSH supplementation in GSH rich tissues. Low activities of these enzymes were observed in BSO treated GSH deficient tissues. TCB injection markedly induced hepatic and renal CYP1A catalytic (ethoxyresorufin O-deethylase [EROD]) activities. This effect was further potentiated (3-fold) in GSH-supplemented fish tissues. In contrast, EROD induction by TCB was markedly suppressed in GSH deficient (BSO-treated) and lipoate-supplemented fish. The suppression of CYP1A catalytic activities in GSH deficient and lipoate-supplemented fish was consistently associated with a suppression of TCB induced CYP1A mRNA and protein expressions in these groups. In glutathione-supplemented fish, TCB induced CYP1A protein expression was markedly higher following 3 days of GSH supplementation. Results of our study suggest that tissue thiol status modulates cytochrome P450 CYP1A gene expression and catalytic activity.

Induction of cytochrome P450 3A by retinoids in rat hepatocyte culture.

1. Rat hepatocytes cultured on a Matrigel matrix were exposed for 48 h to all-trans-retinoic acid, 9-cis-retinoic acid, 13-cis-retinoic acid or fenretinide. 2. Cytochrome P450 3A (CYP3A) RNA levels were increased by approximately eightfold in hepatocytes treated with the retinoids compared to control cultures. 3. CYP1A1 and CYP1A2 RNA levels were only slightly affected or unaffected by the retinoids. 4. The induction of CYP3A by these therapeutically-useful retinoids suggests that they may share a common mechanism for accelerated drug catabolism and acquired clinical resistance.

CYP2D6-related oxidation polymorphism in a Canadian Inuit population.

The xenobiotic oxidation polymorphism associated with cytochrome P450 2D6 (CYP2D6) was investigated in 152 genetically related and unrelated healthy Inuit subjects living in the High Arctic of eastern Canada. Phenotyping was based on HPLC determination of the CYP2D6-related dextromethorphan metabolic ratio in overnight urine samples after oral administration of 30 mg dextromethorphan hydrobromide. The log metabolic ratio was bimodally distributed, with three subjects classified as poor metabolizers (PMs). In subjects unrelated in the first degree, the incidence of the PM phenotype was 3 of 90 or 3.3%. PCR-based analyses of DNA for variants of the CYP2D6 gene demonstrated that the PMs of dextromethorphan had the defective allele CYP2D6*4. The estimated frequency of the CYP2D6*4 allele was 0.067-0.083, which is lower than the frequency in Caucasians but higher than the frequency in Oriental populations. The CYP2D6*3 and the CYP2D6*6 alleles were not detected in the Inuit population. The CYP2D6*10 allele was present in only four unrelated subjects, classified as extensive metabolizers (EMs), resulting in an estimated allele frequency of 0.022, which is much lower than in Oriental populations. This study demonstrated the existence of the CYP2D6 polymorphism in Canadian Inuit, while the frequencies of allelic variants of CYP2D6 point to the uniqueness of this population. Several important therapeutic drugs that are being prescribed in Arctic communities will have altered pharmacokinetics in PMs of CYP2D6.

Differences in caffeine 3-demethylation activity among inbred mouse strains: a comparison of hepatic Cyp1a2 gene expression between two inbred strains.

The 3-demethylation of caffeine can be used as an index of cytochrome P450 CYP1A2 activity in vivo. We compared the plasma levels of caffeine and the 3-demethylated metabolite. 1,7-dimethylxanthine, in six common inbred strains (A/J, P/J, BALB/cJ, C3H/HeJ, AKR/J, and SWR/J) and one inbred strain (APN) derived in our laboratory from outbred Swiss-Webster mice on the basis of its relative susceptibility to acetaminophen-induced hepatotoxicity. We found significant variations between a number of the common strains, all of which produced significantly higher caffeine 3-demethylation indices than our APN strain. In three of the six common strains, there was a significant difference between males and females, with the females having consistently lower 1,7-xanthine/caffeine ratios. Hepatic Cyp1a2 expression was compared between APN and C3H/HeJ males. Microsomal methoxyresorufin O-demethylation, acetanilide 4-hydroxylation, and CYP1A2 immunoreactive protein levels were significantly higher in C3H/HeJ relative to APN mice, as were hepatic CYP1A2 mRNA levels. These results indicate the importance of strain and gender to the outcome of pharmacological or toxicological studies involving CYP1A2-mediated metabolism, as well as the suitability of the plasma 1,7-dimethylxanthine/caffeine ratio as a marker of CYP1A2 activity in the mouse. The striking differences observed between the APN and C3H/HeJ mice suggest that these strains may be suitable for a genetic analysis of the regulation of the basal expression of CYP1A2, a key enzyme in procarcinogen activation.

Glutathione regulates 3,3',4,4'-tetrachlorobiphenyl induced cytochrome P450 metabolism: evidence for a cross-talk between the two major detoxication pathways.

The cytochrome P450 and the glutathione systems are two major pathways of xenobiotic metabolism. We tested the effect of hepatic glutathione content on P450 CYP1A1/2 induction by 3,3',4,4'-tetrachlorobiphenyl in rainbow trout (Oncorhynchus mykiss). Hepatic glutathione status of tetrachlorobiphenyl injected fish could be successfully manipulated by injecting (i.p.) glutathione or L-buthionine-[S,R]-sulfoximine to arrest glutathione synthesis. Tetrachlorobiphenyl injection resulted in a 17-fold increase in CYP1A catalytic (ethoxyresorufin O-deethylase [EROD]) activity. This effect was further potentiated by 2.7-fold in fish in which hepatic glutathione content was elevated by 3.6-fold. The induction of EROD activity by tetrachlorobiphenyl was 7-fold lower in glutathione deficient (78%) liver. Hepatic glutathione deficiency also downregulated tetrachlorobiphenyl-induced CYP1A gene expression as indicated by lower CYP1A RNA levels. Elevated hepatic glutathione did not influence tetrachlorobiphenyl-induced CYP1A RNA level, but enhanced CYP1A protein expression. These enzyme activity, RNA and protein expression data present compelling evidence suggesting the involvement of tissue glutathione in the regulation of tetrachlorobiphenyl-induced cytochrome P450 dependent metabolism.

Induction of CYP3A and associated terfenadine N-dealkylation in rat hepatocytes cocultured with 3T3 cells.

Long-term culture of hepatocytes has been challenged by the loss of differentiated functions. In particular, there is a rapid decline in cytochrome P450 (CYP). In this study, we cocultured rat hepatocytes with 3T3 fibroblasts for 10 days, and examined hepatocyte viability, morphology, and expression of CYP3A. Terfenadine was incubated with the cultures, and its biotransformation was quantitatively analyzed by HPLC. Terfenadine is metabolized by two major pathways: C-hydroxylation to an alcohol metabolite which is further oxidized to a carboxylic acid, and N-dealkylation to azacyclonol. In rat liver, only the N-dealkylation pathway appears to be mediated by CYP3A since anti-rat CYP3A antibody inhibited azacyclonol but not alcohol metabolite formation in incubations of terfenadine with liver microsomes. Freshly isolated rat hepatocytes were seeded on top of confluent 3T3 cells. Cultures were maintained in Williams' E medium supplemented with 10% fetal bovine serum and either 0.1 mumol/L or 5 mumol/L dexamethasone. In pure hepatocyte cultures, viability, as determined by lactate dehydrogenase (LDH) activity, decreased steadily to less than 30% of initial levels by day 10. In cocultures, LDH activity remained high and was 70% of initial levels on day 10. The half-life of terfenadine disappearance was optimally maintained in cocultures treated with 5 mumol/L dexamethasone, and was associated with the increased formation of azacyclonol. On day 5, nearly 50% of added 5 mumol/L terfenadine was converted to azacyclonol within 6 h, whereas the conversion was only 4% on day 1. Western and RNA-slot blot analyses confirmed that treatment with 5 mumol/L dexamethasone induced CYP3A mRNA expression and CYP3A protein expression. This coculture system could offer a useful approach in the study of drugs and xenobiotics metabolized by CYP3A.

Exclusion of linkage between hypokalemic periodic paralysis (HOKPP) and three candidate loci.

Hypokalemic periodic paralysis (HOKPP) is an autosomal dominant neuromuscular disorder characterized by flaccid paralysis accompanied by lowered serum potassium levels. We have tested polymorphic markers linked to the adult skeletal muscle sodium channel (SCN4A) locus at 17q23-q25, the T-cell receptor beta (TCRB) locus at 7q35, and the H-Ras cellular proton-cogene locus (HRAS) at 11p15.5 for linkage with the affected phenotype in a single multigenerational pedigree. No evidence for genetic linkage to HOKPP was found at any of the candidate loci.

Linkage of Thomsen disease to the T-cell-receptor beta (TCRB) locus on chromosome 7q35.

The chromosomal localization of the gene for Thomsen disease, an autosomal dominant form of myotonia congenita, is unknown. Electrophysiologic data in Thomsen disease point to defects in muscle-membrane ion-channel function. A mouse model of myotonia congenita appears to result from transposon inactivation of a muscle chloride-channel gene which maps to a region of mouse chromosome 6. The linkage group containing this gene includes several loci which have human homologues on human chromosome 7q31-35 (synteny), and this is a candidate region for the Thomsen disease locus. Linkage analysis of Thomsen disease to the T-cell-receptor beta (TCRB) locus at 7q35 was carried out in four pedigrees (25 affected and 23 unaffected individuals) by using a PCR-based dinucleotide repeat polymorphism in the TCRB gene. Two-point linkage analysis between Thomsen disease and TCRB showed a maximum cumulative lod score of 3.963 at a recombination fraction of .10 (1-lod support interval .048-.275). We conclude that the Thomsen disease locus is linked to the TCRB locus in these families.

Linkage analysis of candidate loci in autosomal dominant myotonia congenita.

Electrophysiologic studies in patients with autosomal dominant myotonia congenita (ADMC) have implicated defects of both muscle membrane sodium and chloride channels. An adult skeletal muscle sodium channel (ASkM1) gene maps to chromosome 17q23-25, and defects in this gene are almost certainly responsible for at least three variants of hyperkalemic periodic paralysis (HPP)--myotonic HPP, nonmyotonic HPP, and paramyotonia congenita. A gene for a muscle chloride channel has not yet been mapped in humans, but has been identified in the mouse. The gene for the cystic fibrosis transmembrane regulator (CFTR), which has chloride channel properties, is located on chromosome 7q31. This region is syntenic with the area of mouse chromosome 6 that contains the muscle chloride channel gene, a defect in which is responsible for the ADR phenotype, a murine model of myotonia. We performed linkage analysis using chromosome 17q polymorphisms at D17S74, SCN4A, and GH1, two chromosome 7q31 restriction fragment length polymorphisms, and a dinucleotide repeat polymorphism within the CFTR gene (CFTR-DNR), in three pedigrees with ADMC. The lod scores obtained show that the locus for ADMC is not at ASkM1 and is excluded from a region of at least 24 cM on either side of the CFTR gene.

Temporal mapping of the differentiation pathway of the murine erythroleukemia cell.

These studies are concerned with "mapping" the temporal order of the precommitment events in the differentiation pathway of the Friend erythroleukemia cell. We have used a single-block procedure in which a differentiation-specific inhibitor of a temperature-sensitive (ts) differentiation-defective mutation was used to block the differentiation program. Later, the block was removed, differentiation was allowed to proceed, and the time required to reach a reference marker was monitored. These studies have indicated that the mutations tsC2GP1 and tsB5A, the poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide, and the glucocorticoid hormone dexamethasone blocked functions which are required just prior to commitment. We have also used a double-block procedure involving two consecutive restrictive conditions, which suggests that the 3-aminobenzamide- and tsC2GP1-blocked functions constitute a part of a sequentially ordered pathway leading to terminal differentiation. The convergence of the 3-aminobenzamide, dexamethasone, and ts mutational blocks just prior to commitment suggests that the blocked functions may be part of a major control mechanism for commitment. In these studies, we have introduced a cytochalasin B-based assay to monitor commitment. The use of cytochalasin B permits a direct assay for commitment and obviates the need for colony-forming assays using semisolid medium, which have inherent problems such as efficiency of plating.