Quantitative trait locus mapping of genes regulating pulmonary PKC activity and PKC-alpha content.

Strain A/J mice, which are predisposed to experimentally induced asthma and adenocarcinoma, have the lowest pulmonary protein kinase (PK) C activity and content among 22 inbred mouse strains. PKC in neonatal A/J mice is similar to that in other strains, so this difference reflects strain-dependent postnatal regulation. PKC activity is 60% higher in C57BL/6J (B6) than in A/J lungs, and the protein and mRNA concentrations of PKC-alpha, the major pulmonary PKC isozyme, are two- to threefold higher in B6 mice. These differences result from more than a single gene as assessed in F(1), F(2), and backcross progeny of B6 and A/J parents. Quantitative trait locus (QTL) analysis of 23 AxB and BxA recombinant inbred strains derived from B6 and A/J progenitors indicates a major locus regulating lung PKC-alpha content that maps near the Pkcalpha structural gene on chromosome 11 (D11MIT333; likelihood ratio statistic = 12.5) and a major locus controlling PKC activity that maps on chromosome 3 (D3MIT19; likelihood ratio statistic = 15.4). The chromosome 11 QTL responsible for low PKC-alpha content falls within QTLs for susceptibilities to lung tumorigenesis and ozone-induced toxicity.

Cholesteryl ester transfer protein corrects dysfunctional high density lipoproteins and reduces aortic atherosclerosis in lecithin cholesterol acyltransferase transgenic mice.

Expression of human lecithin cholesterol acyltransferase (LCAT) in mice (LCAT-Tg) leads to increased high density lipoprotein (HDL) cholesterol levels but paradoxically, enhanced atherosclerosis. We have hypothesized that the absence of cholesteryl ester transfer protein (CETP) in LCAT-Tg mice facilitates the accumulation of dysfunctional HDL leading to impaired reverse cholesterol transport and the development of a pro-atherogenic state. To test this hypothesis we cross-bred LCAT-Tg with CETP-Tg mice. On both regular chow and high fat, high cholesterol diets, expression of CETP in LCAT-Tg mice reduced total cholesterol (-39% and -13%, respectively; p < 0.05), reflecting a decrease in HDL cholesterol levels. CETP normalized both the plasma clearance of [(3)H]cholesteryl esters ([(3)H]CE) from HDL (fractional catabolic rate in days(-1): LCAT-Tg = 3.7 +/- 0.34, LCATxCETP-Tg = 6.1 +/- 0.16, and controls = 6.4 +/- 0.16) as well as the liver uptake of [(3)H]CE from HDL (LCAT-Tg = 36%, LCATxCETP-Tg = 65%, and controls = 63%) in LCAT-Tg mice. On the pro-atherogenic diet the mean aortic lesion area was reduced by 41% in LCATxCETP-Tg (21.2 +/- 2.0 micrometer(2) x 10(3)) compared with LCAT-Tg mice (35.7 +/- 2.0 micrometer(2) x 10(3); p < 0.001). Adenovirus-mediated expression of scavenger receptor class B (SR-BI) failed to normalize the plasma clearance and liver uptake of [(3)H]CE from LCAT-Tg HDL. Thus, the ability of SR-BI to facilitate the selective uptake of CE from LCAT-Tg HDL is impaired, indicating a potential mechanism leading to impaired reverse cholesterol transport and atherosclerosis in these animals. We conclude that CETP expression reduces atherosclerosis in LCAT-Tg mice by restoring the functional properties of LCAT-Tg mouse HDL and promoting the hepatic uptake of HDL-CE. These findings provide definitive in vivo evidence supporting the proposed anti-atherogenic role of CETP in facilitating HDL-mediated reverse cholesterol transport and demonstrate that CETP expression is beneficial in pro-atherogenic states that result from impaired reverse cholesterol transport.

Additional evidence that the K-ras protooncogene is a candidate for the major mouse pulmonary adenoma susceptibility (Pas-1) gene.

A locus for mouse pulmonary adenoma susceptibility, Pas-1, has been mapped on distal chromosome 6, where the K-ras gene is located. Allele-specific activation and expression of the K-ras allele from the susceptible parent has been observed in lung tumors from F1 hybrid mice. We report here genetic mapping of lung tumor susceptibility genes in urethane-treated A x B and B x A recombinant inbred (RI) mice using microsatellite markers to seek further evidence for the K-ras gene as candidate for Pas-1. The K-ras genotype differs between the A/J and C57BL/6J progenitors of the RI strains, and distal chromosome 6 contained a major lung tumor susceptibility determinant in the RI mice. Additional evidence that Pas-1 is K-ras involved linkage analysis of (A/JOLaHsd x BALB/ cOLaHsd) F2 intercross mice whose parents shared the same K-ras genotype. In contrast to the results with the A x B and B x A RI strains, no distal chromosome 6 site was significantly associated with tumor development in these F2 mice. In addition to this major locus, linkage analysis of the RI mice revealed additional quantitative trait loci for susceptibility on chromosomes 10, 17, and 19. These loci may serve as modifiers of Pas-1. The relationship between the K-ras genotype and the frequency of K-ras mutations in urethane-induced lung tumors from the RI mice was also explored. All 18 tumor DNAs from RI mice with high susceptibility contained an AT-->TA transversion at the second base of K-ras codon 61. This was also true for DNAs from 27 of 27 (100%) tumors in mice with high intermediate susceptibility. In RI strains with a low intermediate susceptibility, the DNA from 39 of 47 (83%) tumors contained an AT-->TA transversion at codon 61, and only 13 of 21 (62%) tumors had this mutation in the most resistant group. This reflects a positive correlation between the frequency of K-ras mutations in lung tumors of A x B or B x A RI strains and their susceptibility to lung carcinogenesis. Since K-ras appears to be Pas-1, these results suggest that some RI mice that have the resistant K-ras or Pas-1 allele undergo tumor development by a K-ras-independent route.

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 7 to X.

The SWXJ recombinant inbred (RI) set was developed for genetic analysis of heritable ovarian tumors. In this report we present data for 223 simple sequence length polymorphisms spanning Chromosomes (Chrs) 7-X to complete the genetic marking of this RI set. The strain distribution patterns (SDP) for these loci were combined with data from 19 other polymorphic genes, resulting in densely marked maps for Chrs 7-X. Combined with the 165 loci for Chr 1-6 reported previously (Svenson et al., Mamm. Genome 6, 867, 1995), the SWXJ RI set represents a powerful tool for mapping genes in neoplastic as well as other heritable disorders.

Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors.

Apolipoprotein E (apoE)-deficient mice develop marked hyperlipidemia as well as atherosclerosis and thus are an excellent animal model for evaluating the potential for gene therapy in human genetic dyslipoproteinemias. Recombinant adenovirus containing either human apoE (rAdv.apoE) or the reporter gene luciferase (rAdv.luc) were generated and infused intravenously in apoE-deficient mice with preinfusion plasma total cholesterol of 644 +/- 149 mg/dl an cholesterol rich VLDL/IDL. After a single infusion of rAdv.apoE, plasma concentrations of human apoE ranging from 1.5 to 650 mg/dl were achieved. Adenovirus-mediated apoE replacement resulted in normalization of the lipid and lipoprotein profile with markedly decreased total cholesterol (103 +/- 18mg/dl), VLDL, IDL, and LDL, as well as increased HDL. Measurement of aortic atherosclerosis 1 mo after adenoviral infusion demonstrated a marked reduction in the mean lesion area of mice infused with rAdv.apoE (58 +/- 8 x 10(3) microns2) when compared with control mice infused with rAdv.luc (161 +/- 10 x 10(3) microns2; P < 0.0001). Thus, apoE expression for 4 wk was sufficient to markedly reduce atherosclerosis, demonstrating the feasibility of gene therapy for correction of genetic hyperlipidemias resulting in atherosclerosis. The combined use of adenovirus vectors and the apoE-deficient mouse represents a new in vivo approach that will permit rapid screening of candidate genes for the prevention of atherosclerosis.

Mouse apolipoprotein J: characterization of a gene implicated in atherosclerosis.

Apolipoprotein J (apoJ), a glycoprotein associated with subclasses of plasma high density lipoproteins (HDL), was found to accumulate in aortic lesions in a human subject with transplantation-associated arteriosclerosis and in mice fed a high-fat atherogenic diet. Foam cells present in mouse aortic valve lesions expressed apoJ mRNA, suggesting local synthesis contributes to apoJ's localization in atherosclerotic plaque. As a prerequisite for elucidating the physiological function of apoJ by using a mouse model, cDNA clones representing the mouse homolog of apoJ were isolated, characterized, and sequenced. The nucleotide sequence predicts a 448 amino acid, 50,260 dalton protein. There was 81% nucleotide sequence similarity between mouse and human apoJ, and 75% similarity at the amino acid level. Mouse apoJ contains six potential N-glycosylation sites, a potential Arg-Ser cleavage site to generate alpha and beta subunits, a cluster of five cysteine residues in each subunit, three putative amphipathic helices, and four potential heparin-binding domains. Southern blot analysis indicates that the gene encompasses approximately 23 kb of DNA. Recombinant inbred strains were used to map apoJ to mouse chromosome 14, tightly linked to Mtv-11. All of the transcribed portions of the gene were cloned and analyzed, and all intron-exon boundaries were defined. The first of the 9 exons is untranslated. Single exons encode the signal peptide, the cysteine-rich domain in the alpha subunit, two potential amphipathic helices flanking a heparin-binding consensus sequence, and a potential amphipathic helix overlapping a heparin-binding domain, supporting their potential functional significance in apoJ. A variety of mouse tissues constitutively express a 1.9 kb apoJ mRNA, with apparently identical transcriptional start sites utilized in all tissues tested. ApoJ mRNA was most abundant in stomach, liver, brain, and testis, with intermediate levels in heart, ovary, and kidney. The high degree of similarity between mouse and human apoJ, in structure and distribution of the gene product, gene structure, and deposition in atherosclerotic plaques, suggests that the mouse is an ideal model with which to elucidate the role of apoJ in HDL metabolism and atherogenesis.

The AXB and BXA set of recombinant inbred mouse strains.

The recombinant inbred (RI) set of strains, AXB and BXA, derived from C57BL/6J and A/J, originally constructed and maintained at the University of California/San Diego, have been imported into The Jackson Laboratory and are now in the 29th to 59th generation of brother-sister matings. Genetic quality control testing with 45 proviral and 11 biochemical markers previously typed in this RI set indicated that five strains had been genetically contaminated sometime in the past, so these strains have been discarded. The correct and complete strain distribution patterns for 56 genetic markers are reported for the remaining RI strain set, which consists of 31 living strains and 8 extinct strains for which DNA is available. Two additional strains, AXB 12 and BXA 17, are living and may be added to the set pending further tests of genetic purity. The progenitors of this RI set differ in susceptibility to 27 infectious diseases as well as atherosclerosis, obesity, diabetes, cancer, cleft palate, and hydrocephalus. Thus, the AXB and BXA set of RI strains will be useful in the genetic analysis of several complex diseases.

Effect of 3-methylcholanthrene on atherosclerosis in two congenic strains of mice with different susceptibilities to methylcholanthrene-induced tumors.

Inbred mouse strains AKXL-38 and AKXL-38a are congenic strains that differ at the Ah locus, a gene which affects the inducibility of the cytochrome P-450 enzymes. The Ah-responsive strain, AKXL-38a, is more susceptible to 3-methylcholanthrene-induced tumors than the Ah-nonresponsive strain, AKXL-38. We previously reported that 3-methylcholanthrene (MC) increased the number and the size of atherosclerotic lesions in a dose-dependent fashion. We now demonstrate that the effect of MC is greater in Ah-responsive mice than in Ah-nonresponsive mice indicating that Ah-responsive mice not only are more susceptible to MC-induced cancer but also are more susceptible to MC-enhanced atherosclerosis. Mice that received atherogenic diet for 14 weeks but no MC had 1.3-1.4 lesions/mouse regardless of genetic type. When mice were treated with MC, the number of lesions increased to 2.1 +/- 0.1 (SE) in Ah-nonresponsive mice, 2.6 +/- 0.2 in Ah-responsive mice, and 2.3 +/- 0.2 in the F1 hybrid. The total area involved in lesions was 9.3-12.6 micron2 in untreated animals. When mice were treated with MC, the total lesion area increased to 23.5 +/- 5.2 micron2 in Ah-nonresponsive mice, to 43.9 +/- 6.6 micron2 in Ah-responsive mice, and to 36.2 +/- 4.8 micron2 in F1 hybrids. Thus MC increased the lesion area in both strains of mice, but the increase was significantly greater in Ah-responsive than in Ah-nonresponsive animals. High density lipoprotein levels were not significantly affected by MC treatment or Ah genotype. In order to determine whether the increased susceptibility to MC-induced atherosclerosis segregated with the Ah gene, AKXL-38 and AKXL-38a mice were mated and the F1 progeny were backcrossed to the Ah-nonresponsive parent. Backcross progeny were tested for Ah genotype by zoxazolamine sleeping time. Measurements of lesions showed that increased susceptibility to MC-enhanced atherosclerosis segregated with the Ah locus.

Effect of 3-methylcholanthrene on the development of aortic lesions in mice.

The effect of a carcinogen, 3-methylcholanthrene (3-MC), on the formation and growth of atherosclerotic lesions in mice was examined. Increasing doses of 3-MC from 15 to 1500 micrograms/kg increased the number and size of lipid-staining lesions in the aorta of AKXL-38a mice that were fed an atherogenic diet for 8 weeks. The number of lesions per mouse was 0.85 +/- 0.19 (SE) for animals treated with 3-MC (150 micrograms/kg) compared to 0.10 +/- 0.10 lesions/mouse for animals given solvent rather than 3-MC. The progression of lesions over time from 5 to 18 weeks showed that 3-MC-treated mice also differed from controls in the size of lesions. The total score per mouse at 18 weeks of atherogenic diet, based on the number of lesions and the size of each lesion, indicated by a score of 1 to 4, was 4.31 +/- 0.71 for 3-MC-treated animals and 2.67 +/- 0.74 for animals given solvent. The effect of 3-MC treatment could be observed at 18 weeks even though the entire dose of 3-MC was given during the first week on the atherogenic diet. These experiments do not distinguish whether 3-MC affects atherosclerotic lesions by acting as a mutagen or by some other mechanism. The composition of an atherogenic diet that produces lesions in mice without high mortality is given as well as a comparison of different methods of evaluating lesion formation.

Theophylline disposition in residents living near a chemical waste site.

The pharmacokinetics of theophylline were compared in 11 residents living near a chemical waste site (Love Canal) and 25 control subjects from Western New York. Love Canal residents had documented chronic exposure to a variety of organic and halogenated compounds. Only young adults with no overt diseases were examined. Theophylline plasma clearances were similar in the two groups, although the enzyme inductive effects of tobacco smoking were further confirmed. The lack of environmental effect on theophylline clearance indicates that no chronic net change in one pathway of microsomal P-448 oxidation occurred. The possibility of acute changes related to seasonal chemical exposure and mixed inductive/repressive effects from the myriad types of chemicals may confound the data.

Urinary glucuronidase and arylsulfatases in identical twins of bladder cancer patients.

Studies showing that bladder cancer patients have unusually high levels of urinary beta-glucuronidase and arylsulfatases A and B led to the suggestion that these urinary enzymes may participate in bladder cancer etiology. An alternative explanation of the high levels of these urinary enzymes in bladder cancer patients is that the disease itself causes the elevation. Since the levels of these enzymes are genetically determined, measuring these enzymes in healthy identical twins of bladder cancer patients can test whether high enzyme levels occurred prior to bladder cancer. Five healthy identical cotwins of bladder cancer patients, together with matched controls, were measured for urinary beta-glucuronidase, arylsulfatases A and B, and two other lysosomal enzymes as controls, alpha- and beta-galactosidases. The mean levels of all five enzymes were not very different in the cotwins and controls, suggesting that high levels of urinary enzymes observed in bladder cancer patients are a consequence of disease rather than occurring prior to disease and contributing to its etiology.

Role of urinary beta-glucuronidase in human bladder cancer.

It has been suggested that high levels of urinary beta-glucuronidase may increase an individual's risk of bladder cancer by releasing free carcinogens from their inactive glucuronide conjugates in the bladder. The hypothesis derives in part from the high levels of urinary beta-glucuronidase observed in bladder cancer patients. Because most of the individual variation in levels of urinary beta-glucuronidase and other lysosomal enzymes in the normal population is genetically determined, we would expect that, if high glucuronidase levels were a predisposing factor in the disease, bladder cancer patients would transmit this trait to their progeny. We have tested this hypothesis and find that levels of urinary beta-glucuronidase and three other lysosomal enzymes, alpha-galactosidase, beta-galactosidase, and beta-hexosaminidase, are not significantly elevated in 34 progeny of bladder cancer patients compared to 34 matched controls. Additionally, 15 bladder cancer patients judged to be disease free for a median time of 5 years did not have elevated levels of urinary beta-glucuronidase when compared to a normal population of 125 individuals. Thus, the high levels of glucuronidase observed in bladder cancer patients are most likely a consequence of disease rather than a cause.

Controversy at Love Canal.

KIE: A cancer researcher reviews the events surrounding the toxic waste contamination at Love Canal with emphasis on the political nature of the controversy about its health impact. Antagonism between the community and the New York State Department of Health was fueled by several factors: the state's awareness that it gained from delay in investigation, disagreement on health problems to be studied, control over the information gathering process, silencing of opposition opinion, and the violation of norms of scientific behavior. The author calls for the establishment of standards of ethical behavior for scientists in such situations, standards for conflict resolution, and means of appeal for those injured.^ieng

Absence of seasonal variation in antipyrine metabolism.

The induced activity of aryl hydrocarbon hydroxylase (AHH), measured by the metabolism of benzo[a]pyrene to fluorescent products in cultured human lymphocytes, shows a strong seasonal variation. The in vivo metabolism of antipyrine, which is also catalyzed by microsomal cytochrome P-450-dependent monooxygenases, has been reported to be correlated with AHH inducibility in human lymphocytes. To determine whether antipyrine metabolism also showed seasonal changes, we measured antipyrine half-life (t 1/2) in 10 nonsmokers and eight smokers at the two times of the year that correspond to the high and low peaks of inducible AHH activity as measured in lymphocytes. The mean antipyrine t 1/2 determined in all 18 subjects in summer was almost identical to that found in winter (mean +/- SEM = 10.90 +/- 0.65 and 10.96 +/- 0.78 hr). AHH activity in cultured human lymphocytes from the nonsmoking subjects was determined in control and 3-methylcholanthrene-induced cells to obtained inducibility ratios of 4.2 +/- 0.56 (SEM) in the summer and 1.4 +/- 0.14 (SEM) in winter. These results indicate that the seasonal variation in AHH inducibility in human lymphocytes is not reflected by a corresponding seasonal variation in antipyrine metabolism in vivo.

Seasonal variation of aryl hydrocarbon hydroxylase activity in human lymphocytes.

A seasonal variation was observed when aryl hydrocarbon hydroxylase (AHH) activity was measured in the cultured lymphocytes of 977 donors over a period of 2 1/2 years. The variation was strongest in AHH activity induced by 3-methylcholanthrene and was less apparent for AHH activity in lymphocytes grown without any inducer. The period of the seasonal variation is 1 year, and maximal induced AHH activity occurs during late summer and early fall with minimal activity 6 months later. Based on the average of all individuals tested during the highest and lowest weeks, induced AHH activity can be as much as 10-fold higher during the peak season. It is not possible to tell from these experiments whether the seasonal variation is tissue specific, occurring only in lymphocytes, or characteristic of microsomal oxidases in other tissues as well.

High-pressure liquid chromatographic analysis of benzo(a)pyrene metabolism by human lymphocytes from donors of different aryl hydrocarbon hydroxylase inducibility and antipyrine half-lives.

With high-pressure liquid chromatography (HPLC), lymphocytes from six human donors were evaluated for their ability to metabolize benzo(a)pyrene (BP). Donors whose aryl hydrocarbon hydroxylase (AHH) inducibility ratios ranged from 2.4 to 4.6 and whose antipyrine plasma half-lives ranged from 8 to 17 hr were examined. The BP metabolites identified were: 7,8-dihydrodiol, quinones, and 9-hydroxy and 3-hydroxy phenols. HPLC profiles of BP metabolites elaborated by uninduced (control) and benz(a)anthracene-induced lymphocytes were qualitatively similar among the six donors. A good correlation (r = 0.79) was found between known AHH inducibility ratios for the donors, as determined by the conventional fluorometric AHH assay, and induction of BP phenol production quantitated from HPLC data. HPLC results also indicated that the induction of benzo(a)pyrene-7,8-dihydrodiol, the proposed proximate carcinogenic form of BP, did not parallel BP phenol induction. Furthermore, the data also indicated a good negative correlation between AHH inducibility and the measurements of plasma antipyrine or urinary 4-hydroxyantipyrine half-lives (r = -0.88 or -0.91), respectively.

Aryl hydrocarbon hydroxylase inducibility is not altered in bladder cancer patients or their progeny.

We investigated the possible influence of aryl hydrocarbon hydroxylase (AHH) on susceptibility to bladder cancer in humans. AHH inducibility was measured in the cultured lymphocytes of 16 patients who were being followed after successful treatment for bladder cancer, in 53 progeny of bladder cancer patients, and in matched controls. In both the progeny and patient populations, no evidence was found for a difference between the distribution of AHH inducibility or induced AHH activity compared to the distribution among control individuals. Thus, AHH acitivity or inducibility does not appear to be a major determinant of bladder cancer risk in humans.

Comparison of benzo[a]pyrene metabolism by human peripheral blood lymphocytes and monocytes.

Here we report a more sensitive high-pressure liquid chromatographic (HPLC) assay of benzo[a]pyrene (BP) metabolites elaborated by human monocytes and lymphocytes using only 10 X 10(6) cells for HPLC analysis. The major metabolites formed by both lymphocytes and monocytes were 3-hydroxy-BP, 9-hydroxy-BP and quinones. BP-dihydrodiols were also found by HPLC analysis, the major one being BP-7,8-dihydrodiol for both cell types. In addition, a peak slightly more polar than 9,10-dihydrodiol was formed by lymphocytes. The 7,8-diol peak was eliminated by the addition of 1,1,1-trichloropropene oxide to the incubation mixture. The presence of alpha-napthoflavone resulted in an overall decrease in metabolite production.