Outcome of patients with metastatic renal cell carcinoma treated with targeted therapy without cytoreductive nephrectomy.

BACKGROUND: Cytoreductive nephrectomy (CN) became a standard procedure in metastatic renal cell carcinoma (mRCC) in the immunotherapy era. Historically, median overall survival (OS) of patients treated with interferon alpha (IFN-α) without CN was 7.8 months. Median OS in patients treated with targeted therapy (TT) without CN is unknown. PATIENTS AND METHODS: We retrospectively reviewed records of patients with mRCC who received TT without CN. Kaplan-Meier methods and Cox regression analysis were used to estimate median OS and identify poor prognostic factors. RESULTS: One hundred and eighty-eight patients were identified. Most patients had intermediate-risk (54.8%) or poor-risk (44.1%) disease. Median OS for all patients was 10.4 months [95% confidence interval (CI) 8.1-12.5]. By multivariable analysis, elevated baseline lactate dehydrogenase and corrected calcium, performance status of two or more, retroperitoneal nodal metastasis, thrombocytosis, current smoking, two or more metastatic sites, and lymphopenia were independent risk factors for inferior OS. Patients with four or more factors had increased risk of death (hazard ratio 8.83, 95% CI 5.02-15.5, P < 0.001) and 5.5-month median OS. Nineteen patients (10.0%) survived for 2+ years. CONCLUSIONS: These data highlight the improved OS of patients with mRCC treated with TT without CN, compared with historical IFN-α treatment, and may guide the design of trials investigating the role of CN in the TT era.

Gene expression profiles and genetic damage in benzo(a)pyrene diol epoxide-exposed TK6 cells.

Microarray analysis is a powerful tool to identify the biological effects of drugs or chemicals on cellular gene expression. In this study, we compare the relationships between traditional measures of genetic toxicology and mutagen-induced alterations in gene expression profiles. TK6 cells were incubated with 0.01, 0.1, or 1.0 microM +/-anti-benzo(a)pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE) for 4 h and then cultured for an additional 20 h. Aliquots of the exposed cells were removed at 4 and 24 h in order to quantify DNA adduct levels by 32P post-labeling and measure cell viability by cloning efficiency and flow cytometry. Gene expression profiles were developed by extracting total RNA from the control and exposed cells at 4 and 24 h, labeling with Cy3 or Cy5 and hybridizing to a human 350 gene array. Mutant frequencies in the Thymidine Kinase and Hypoxanthine Phosphoribosyl Transferase genes were also determined. The 10alpha-(deoxyguanosin-N(2)-yl)-7alpha,8beta,9beta-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene (dG-N(2)-BPDE) adduct increased as a function of dose and was the only adduct identified. A dose-related decrease in cell viability was evident at 24 h, but not at 4 h. Cell death occurred by apoptosis. At 4 h, analysis of the gene expression profiles revealed that Glutathione Peroxidase and Gadd45 were consistently upregulated (greater than 1.5-fold and significantly (P < 0.001) greater than the control in two experiments) in response to 1.0 microM BPDE exposure. Fifteen genes were consistently down-regulated (less than 0.67-fold and significantly (P < 0.001) lower than the control in two experiments) at 4 h in cultures exposed to 1.0 microM BPDE. Genes with altered expression at 4 h included genes important in the progression of the cell-cycle and those that inhibit apoptosis. At 24 h post-exposure, 16 genes, involved in cell-cycle control, detoxification, and apoptosis were consistently upregulated; 10 genes were repressed in cultures exposed to the high dose of BPDE. Real-time quantitative PCR confirmed the differential expression of selected genes. These data suggest that changes in gene expression will help to identify effects of drugs and chemicals on molecular pathways in cells, and will provide useful information about the molecular responses associated with DNA damage. Of the endpoints evaluated, DNA adduct formation was the most sensitive indicator of DNA damage. DNA adduct formation was clearly evident at low doses, but the number of genes with significantly altered expression (P < 0.001) was minimal. Alterations in gene expression were more robust at doses associated with cellular toxicity and induction of mutations.

Analysis of mutations and bone marrow micronuclei in Big Blue rats fed leucomalachite green.

Leucomalachite green (LMG) is the major metabolite of malachite green (MG), a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over MG and LMG is due to the potential for consumer exposure, suggestive evidence of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. In order to evaluate the risks associated with exposure to LMG, female Big Blue rats were fed up to 543 ppm LMG; groups of these rats were killed after 4, 16, or 32 weeks of exposure and evaluated for genotoxicity. We previously reported that this treatment resulted in a dose-dependent induction of liver DNA adducts, and that the liver lacI mutant frequency (MF) was increased, but only in rats fed 543 ppm LMG for 16 weeks. In the present study, we report the results from lymphocyte Hprt mutant assays and bone marrow micronucleus assays performed on these same rats. In addition, we have determined the types of lacI mutations induced in the rats fed 543 ppm LMG for 16 weeks and the rats fed control diet. No significant increases in the frequency of micronuclei or Hprt mutants were observed for any of the doses or time points assayed. Molecular analysis of 80 liver lacI mutants from rats fed 543 ppm LMG for 16 weeks revealed that 21% (17/80) were clonal in origin and that most (55/63) of the independent mutations were base pair substitutions. The predominant type of mutation was G:C --> A:T transition (31/63) and the majority (68%) of these involved CpG sites. When corrected for clonality, the 16-week lacI mutation frequency (36 +/- 10) x 10(-6) in treated rats was not significantly different from the clonally corrected control frequency (17 +/- 9 x 10(-6); P = 0.06). Furthermore, the lacI mutational spectrum in treated rats was not significantly different from that found for control rats (P = 0.09). Taken together, these data indicate that the DNA adducts produced by LMG in female rats do not result in detectable levels of genotoxicity, and that the increase in lacI MF observed previously in the liver of treated rats may be due to the disproportionate expansion of spontaneous lacI mutations.

Photoreaction, phototoxicity, and photocarcinogenicity of retinoids.

Sunlight is a human carcinogen. Many retinoid-containing cosmetics are used to protect damages caused by sunlight irradiation. Since retinol is thermally unstable and retinyl palmitate (RP) s relatively more stable, RP is also widely used as an ingredient in cosmetic formulations. In general, little is known about the photodecomposition of retinoids and the toxicity of retinoids and their photodecomposition products on the skin's responses to sunlight. This review focuses on the update information on photoreactions, phototoxicity, and photocarcinogenicity of the natural retinoids including retinol, retinal, retinoid acid (RA), retinyl acetate, and RP.

Mutagenicity and carcinogenicity in relation to DNA adduct formation in rats fed leucomalachite green.

Leucomalachite green is a persistent and prevalent metabolite of malachite green, a triphenylmethane dye that has been used widely as an antifungal agent in the fish industry. Concern over the use of malachite green is due to the potential for consumer exposure, evidence suggestive of tumor promotion in rodent liver, and suspicion of carcinogenicity based on structure-activity relationships. Our previous study indicated that feeding rodents malachite or leucomalachite green resulted in a dose-related increase in liver DNA adducts, and that, in general, exposure to leucomalachite green caused an increase in the number and severity of changes greater than was observed following exposure to malachite green. To characterize better the genotoxicity of leucomalachite green, female Big Blue rats were fed leucomalachite green at doses of 0, 9, 27, 91, 272, or 543 ppm for up to 32 weeks. The livers were analyzed for lacI mutations at 4, 16, and 32 weeks and DNA adducts at 4 weeks. Using a 32P-postlabeling assay, we observed a dose-related DNA adduct in the livers of rats fed 91, 272, and 543 ppm leucomalachite green. A approximately 3-fold increase in lacI mutant frequency was found in the livers of rats fed 543 ppm leucomalachite green for 16 weeks, but significant increases in mutant frequencies were not found for any of the other doses or time points assayed. We also conducted 2-year tumorigenesis bioassays in female and male F344 rats using 0, 91, 272, and 543 ppm leucomalachite green. Preliminary results indicate an increasing dose trend in lung adenomas in male rats treated with leucomalachite green, but no increase in the incidence of liver tumors in either sex of rat. These results suggest that the DNA adduct formed in the livers of rats fed leucomalachite green has little mutagenic or carcinogenic consequence.

Suppression of tumorigenicity in the human prostate cancer cell line M12 via microcell-mediated restoration of chromosome 19.

Previously we immortalized human, nontransformed prostate epithelial cells with SV40 large T-antigen (SV40TAg) and derived increasingly aggressive sublines from the immortalized line. The progression of the tumorigenic sublines to metastatic capacity was accompanied by the formation of an unbalanced translocation between chromosomes 16 and 19, resulting in loss of 19p and proximal 19q. To test whether the tumorigenic and/or metastatic phenotype was causally related to this genetic alteration, we restored a neo-tagged human chromosome 19 to M12 cells by microcell-mediated transfer and assessed their growth. In vitro, the resultant hybrids grew more slowly in monolayer culture and showed a significant reduction in anchorage-independent growth as compared to M12neo controls. In vivo, all mice (13/13) injected subcutaneously (SC) with control M12neo cells developed tumors after 9-15 days. In contrast, 9/15 mice injected SC with microcell-transferred chromosome 19 hybrid cells failed to form tumors, with 6/15 producing very small tumors after 120 days. Analysis of three of these six tumors showed consistent, new chromosomal changes. Furthermore, in one of the tumors, loss of a chromosome 19 was noted in 40% of the cells. After intraprostatic injections of the hybrid cells, only 2/7 mice developed microscopic tumors, with no metastases. These data suggest the presence of a gene or genes on chromosome 19 that function to suppress growth.

DNA adduct measurements, cell proliferation and tumor mutation induction in relation to tumor formation in B6C3F1 mice fed coal tar or benzo[a]pyrene.

Coal tar is a complex mixture containing hundreds of compounds, at least 30 of which are polycyclic aromatic hydrocarbons, including the carcinogen benzo[a]pyrene (BaP). Although humans are exposed to complex mixtures on a daily basis, the synergistic or individual effects of components within a mixture on the carcinogenic process remain unclear. We have compared DNA adduct formation and cell proliferation in mice fed coal tar or BaP for 4 weeks with tumor formation in a 2 year chronic feeding study. Additionally, we have analyzed tumor DNA for mutations in the K-ras, H-ras and p53 genes. In the forestomach of mice fed either coal tar or BaP an adduct indicative of BaP was detected, with adduct levels increasing in a dose-responsive manner. K-ras mutations were detected in the forestomach tumors, with the incidence being similar in mice fed coal tar or BaP. These results suggest that the BaP within coal tar is associated with forestomach tumor induction in coal tar-fed mice. DNA adduct levels in the small intestine were not predictive of tumor incidence in this tissue; instead, the tumors appeared to result from compound-induced cell proliferation at high doses of coal tar. K-ras mutations were detected in lung tumors. Since lung tumors were not increased by BaP, coal tar components other than BaP appear to be responsible for the tumors induced in this tissue. H-ras mutations, primarily occurring at codon 61, were the most common mutation observed in liver tumors induced by coal tar. Since this mutation profile is observed in spontaneous hepatic tumors, components in the coal tar may be promoting the expansion of pre-existing lesions.

Cancer risk estimation for mixtures of coal tars and benzo(a)pyrene.

Two-year chronic bioassays were conducted by using B6C3F1 female mice fed several concentrations of two different mixtures of coal tars from manufactured gas waste sites or benzo(a)pyrene (BaP). The purpose of the study was to obtain estimates of cancer potency of coal tar mixtures, by using conventional regulatory methods, for use in manufactured gas waste site remediation. A secondary purpose was to investigate the validity of using the concentration of a single potent carcinogen, in this case benzo(a)pyrene, to estimate the relative risk for a coal tar mixture. The study has shown that BaP dominates the cancer risk when its concentration is greater than 6,300 ppm in the coal tar mixture. In this case the most sensitive tissue site is the forestomach. Using low-dose linear extrapolation, the lifetime cancer risk for humans is estimated to be: Risk < 1.03 x 10(-4) (ppm coal tar in total diet) + 240 x 10(-4) (ppm BaP in total diet), based on forestomach tumors. If the BaP concentration in the coal tar mixture is less than 6,300 ppm, the more likely case, then lung tumors provide the largest estimated upper limit of risk, Risk < 2.55 x 10(-4) (ppm coal tar in total diet), with no contribution of BaP to lung tumors. The upper limit of the cancer potency (slope factor) for lifetime oral exposure to benzo(a)pyrene is 1.2 x 10(-3) per microgram per kg body weight per day from this Good Laboratory Practice (GLP) study compared with the current value of 7.3 x 10(-3) per microgram per kg body weight per day listed in the U.S. EPA Integrated Risk Information System.

DNA adduct formation and molecular analysis of in vivo lacI mutations in the mammary tissue of Big Blue rats treated with 7, 12-dimethylbenz[a]anthracene.

Recently we compared the lacI and Hprt mutant frequencies (MFs) and types of mutations in lymphocytes of Big Blue((R)) (BB) rats exposed to 7,12-dimethylbenz[a]anthracene (DMBA) under conditions that result in mammary gland tumors. In this study, we have examined the target mammary tissue for DMBA-induced DNA adducts, lacI MF and types of lacI mutations. Seven-week-old female BB rats were given single doses of 0, 20 or 130 mg/kg DMBA by gavage and the DNA adducts and lacI MFs in the mammary tissue were measured over a period of 14 days and 18 weeks, respectively, following treatment. The lacI MF in the mammary tissue increased for 10 weeks and then remained relatively constant; 130 mg/kg DMBA produced a 14-fold increase in the MF (255 +/- 50 x 10(-6) p.f.u.) over control MF (18. 3 +/- 4 x 10(-6) p.f.u.). (32)P-post-labeling analysis of DNA from mammary tissue and splenic lymphocytes of treated rats revealed two major adducts. Comparison of these adducts with DMBA standards indicated that the adducts formed by DMBA involved both G:C and A:T base pairs. DNA sequencing revealed that the majority of DMBA-induced lacI mutations were base pair substitutions and that A:T-->T:A (44% of the independent mutations) and G:C-->T:A (24% of the independent mutations) transversions were the predominant types. Furthermore, the mutational results revealed a 'hotspot' for a G-->T mutation in codon 95 (GTG-->TTG) of the lacI gene in mammary tissue. These results suggest that DMBA is highly mutagenic to lacI in mammary tissue and that adducts with both G:C and A:T base pairs participate in forming mutations in DMBA-treated BB rats.

4-Aryl-2-anilinopyrimidines as corticotropin-releasing hormone (CRH) antagonists.

A series of 4-aryl-2-(N-ethylanilino)pyrimidines has been synthesized as corticotropin-releasing hormone (CRH) inhibitors. The effect of substitution on each aromatic ring on receptor binding was investigated.

Use of the Suzuki reaction for the synthesis of aryl-substituted heterocycles as corticotropin-releasing hormone (CRH) antagonists.

The Suzuki reaction has been used to synthesize a variety of aryl-substituted heterocyclic antagonists of the CRH1 receptor. Examples with several different heterocyclic cores are potent CRH receptor ligands.

Non-peptide corticotropin-releasing hormone antagonists: syntheses and structure-activity relationships of 2-anilinopyrimidines and -triazines.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

Toxicity and metabolism of malachite green and leucomalachite green during short-term feeding to Fischer 344 rats and B6C3F1 mice.

Malachite green, an N-methylated diaminotriphenylmethane dye, has been widely used as an antifungal agent in commercial fish hatcheries. Malachite green is reduced to and persists as leucomalachite green in the tissues of fish. Female and male B6C3F1 mice and Fischer 344 rats were fed up to 1200 ppm malachite green or 1160 ppm leucomalachite green for 28 days to determine the toxicity and metabolism of the dyes. Apoptosis in the transitional epithelium of the urinary bladder occurred in all mice fed the highest dose of leucomalachite green. This was not observed with malachite green. Hepatocyte vacuolization was present in rats administered malachite green or leucomalachite green. Rats given leucomalachite green also had apoptotic thyroid follicular epithelial cells. Decreased T4 and increased TSH levels were observed in male rats given leucomalachite green. A comparison of adverse effects suggests that exposure of rats or mice to leucomalachite green causes a greater number of and more severe changes than exposure to malachite green. N-Demethylated and N-oxidized malachite green and leucomalachite green metabolites, including primary arylamines, were detected by high performance liquid chromatography/mass spectrometry in the livers of treated rats. 32P-Postlabeling analyses indicated a single adduct or co-eluting adducts in the liver DNA. These data suggest that malachite green and leucomalachite green are metabolized to primary and secondary arylamines in the tissues of rodents and that these derivatives, following subsequent activation, may be responsible for the adverse effects associated with exposure to malachite green.

Structure-activity studies on 4-substituted-2-anilinopyrimidine corticotropin releasing factor (CRF) antagonists.

Structure-activity studies around the 4-position of 2-anilinopyrimidine corticotropin releasing factor (CRF) antagonists suggest that there is a large lipophilic cavity in the rat CRF receptor, which can accommodate a wide variety of substituents at this position in contrast to the steric constraints observed for other positions on the 2-anilinopyrimidine core. The chemical syntheses and biological activities of 2-anilinopyrimidine CRF antagonists with carbon-linked substituents at the 4-position are reported. Significant improvements in rat pharmacokinetic parameters were achieved relative to those for the lead structure. While the lead compound 1 (rCRF Ki = 44 nM) afforded no detectable rat plasma levels after intraperitoneal (i.p.) or oral (p.o.) dosing, compounds 3-3 (rCRF Ki = 16 nM) and 3-4 (rCRF Ki 59 nM) gave high rat plasma levels at 30 mg/kg (i.p., p.o.) (Cmax = 1389 nM and 8581 nM (i.p.) respectively; Cmax = 113 nM and 988 nM (p.o.), respectively). Furthermore 3-3 and 3-4 had superior bioavailabilities at these doses (59 and 46% (i.p.), respectively; 2 and 10%, (p.o.), respectively).

Tumors and DNA adducts in mice exposed to benzo[a]pyrene and coal tars: implications for risk assessment.

Current methods to estimate the quantitative cancer risk of complex mixtures of polycyclic aromatic hydrocarbons (PAH) such as coal tar assume that overall potency can be derived from knowledge of the concentration of a few carcinogenic components such as benzo[a]pyrene (B[a]P). Genotoxic damage, such as DNA adducts, is thought to be an essential aspect of PAH-induced tumorigenesis and could be a biomarker for exposure useful for estimating risk. However, the role of B[a]P and the relationship of adduct formation in tumorigenesis have not been tested rigorously in models appropriate for human health risk assessment. Therefore, we directly compared tumor induction and adduct formation by B[a]P and coal tars in several experimental protocols, including one broadly accepted and used by regulators. We found that B[a]P content did not account for tumor incidences after exposure to coal tars. DNA adducts were found in both tumors and tumor-free tissue and tumor outcomes were not predicted by either quantitation of total DNA adducts or by the DNA adduct formed by B[a]P. These data suggest that risk assessments based on B[a]P content may not predict accurately risk to human health posed by environmental PAH.

A comparison of the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay.

The tumorigenicity of two coal tar mixtures was compared to that of benzo[a]pyrene after 2 years of feeding. Mixture 1, a composite of coal tar from seven coal gasification plant waste sites, was fed to female B6C3F1 mice (48 mice per group) for 2 years at doses of 0.0, 0.01, 0.03, 0.1, 0.3, 0.6 and 1.0%. Mixture 2, which was composed of coal tar from two of the seven waste sites and another site having a high benzo[a]pyrene content, was fed at doses of 0.0, 0.03, 0.1 and 0.3%. Additional groups of mice were fed 0, 5, 25 and 100 ppm benzo[a]pyrene. The coal tar diets induced a dose-related increase in hepatocellular adenomas and carcinomas, alveolar/bronchiolar adenomas and carcinomas, forestomach squamous epithelial papillomas and carcinomas, small intestine adenocarcinomas, histiocytic sarcomas, hemangiosarcomas in multiple organs and sarcomas. Benzo[a]pyrene treatment resulted in an increased incidence of papillomas and/or carcinomas of the forestomach, esophagus and tongue. A comparison of the results indicated that the benzo[a]pyrene in the coal tar diets could be responsible for the forestomach tumors. In contrast, the lung and liver tumors appeared to be due to other genotoxic components contained within the coal tar mixture, while the small intestine tumors resulted from chemically-induced cell proliferation that occurred at high doses of coal tar.

Immunochemical, 32P-postlabeling, and GC/MS detection of 4-aminobiphenyl-DNA adducts in human peripheral lung in relation to metabolic activation pathways involving pulmonary N-oxidation, conjugation, and peroxidation.

4-Aminobiphenyl (ABP) is a recognized human bladder carcinogen, whose presence in cigarette smoke results in DNA adduct formation in the human urothelium. Since preliminary studies indicated that even higher levels of ABP-DNA adducts may be present in human peripheral lung, we utilized a sensitive immunochemical assay, in combination with 32P-postlabeling, to quantify the major 4-aminobiphenyl (ABP)-DNA adduct, N-(guan-8-yl)-ABP, in surgical samples of peripheral lung tissue from smokers and ex-smokers. No differences in adduct levels were detected between smokers and ex-smokers by immunoassay. In contrast, the 32P-postlabeling method showed statistically significant differences between adduct levels in smokers and ex-smokers; however, a relatively high background of smoking-related adducts chromatograph near the major ABP adducts and may compromise estimation of the level of ABP-DNA adducts in smokers. Furthermore, the levels measured by 32P-postlabeling were 20- to 60-fold lower than that measured by immunoassay. Since 32P-postlabeling may underestimate and immunochemical assays may overestimate adduct levels in the lung, selected samples were also evaluated by GC/MS. The immunochemical and GC/MS data were concordant, leading us to conclude that N-(guan-8-yl)-ABP adducts were not related to smoking status. Since ABP-DNA adduct levels in human lung did not correlate with smoking status as measured by immunoassay and GC/MS, the metabolic activation capacity of human lung microsomes and cytosols was examined to determine if another exposure (e.g., 4-nitrobiphenyl) might be responsible for the adduct. The rates of microsomal ABP N-oxidation were below the limit of detection, which was consistent with a lack of detectable cytochrome P4501A2 in human lung. N-Hydroxy-ABP O-acetyltransferase (but not sulfotransferase) activity was detected in cytosols and comparative measurements of N-acetyltransferase (NAT) using p-aminobenzoic acid and sulfamethazine indicated that NAT1 and NAT2 contributed to this activity. 4-Nitrobiphenyl reductase activity was found in lung microsomes and cytosols, with the reaction yielding ABP and N-hydroxy-ABP. Lung microsomes also demonstrated high peroxidative activation of ABP, benzidine, 4,4'-methylene-bis(2-chloroaniline), 2-aminofluorene, and 2-naphthylamine. The preferred co-oxidant was hydrogen peroxide and the reaction was strongly inhibited by sodium azide but not by indomethacin or eicosatetraynoic acid, which suggested the primary involvement of myeloperoxidase rather than prostaglandin H synthase or lipoxygenase. This was confirmed by immunoinhibition and immunoprecipitation studies using solubilized human lung microsomes and antisera specific for myeloperoxidase. These data suggest that ABP-DNA adducts in human lung result from some environmental exposure to 4-nitrobiphenyl. The bioactivation pathways appear to involve: (1) metabolic reduction to N-hydroxy-ABP and subsequent O-acetylation by NAT1 and/or NAT2; and (2) metabolic reduction to ABP and subsequent peroxidation by myeloperoxidase. The myeloperoxidase activity appears to be the highest peroxidase activity measured in mammalian tissue and is consistent with the presence of neutrophils and polymorphonuclear leukocytes surrounding particulate matter derived from cigarette smoking.

Lymphocyte mutant frequency in relation to DNA adduct formation in rats treated with tumorigenic doses of the mammary gland carcinogen 7,12-dimethylbenz[a]anthracene.

The ability of the rat lymphocyte hprt assay to detect tissue-specific carcinogens was evaluated using 7,12-dimethylbenz[a]anthracene (DMBA) administered under conditions that result in mammary gland tumors. Fifty-day-old female Sprague-Dawley rats were given single doses of 5 and 20 mg/kg DMBA by gavage, and the frequency of 6-thioguanine-resistant (TGr) T-lymphocytes was measured over a period of 21 weeks. A time- and dose-dependent increase in mutant frequency was found, with a maximum frequency found 9-15 weeks after treatment with 20 mg/kg of DMBA. Rats were also dosed with 1, 2.5, 5, 10, 15 and 20 mg/kg of DMBA and assayed for TGr mutant frequency 10 weeks after treatment. A significant linear dose-response was found, with all the DMBA doses resulting in significant increases in mutant frequency. To determine whether or not DMBA-induced mutants in rat lymphocytes reflected the DNA damage in the target tissue, rats were treated with 5 and 20 mg/kg of DMBA and spleen lymphocytes and mammary gland tissue were assayed for DNA adduct formation 1, 3 and 7 days later. A similar pattern of 32P- postlabeled adducts, involving both dG and dA nucleotides, was found in DNA from both the target tissue and the surrogate lymphocytes. Adduct formation was dose responsive in both tissues, with a 2.3- to 4-fold higher concentration in mammary gland as compared with lymphocytes. These results indicate that the rat lymphocyte hprt assay is sensitive to a mammary gland carcinogen and that similar types of DNA adducts are associated with both the lymphocyte mutants and the mammary gland tumors induced by DMBA.

Insulin-dependent reduction in hepatic and splenic contents of interleukin-1 beta in experimental diabetes.

Interleukin-1 beta (IL-1 beta) is a polypeptide produced by a variety of cells of hematological, dermal and neural origin. We have investigated the effect of type I diabetes mellitus and insulin treatment on tissue levels of IL-1 beta using streptozotocin (STZ)-treated mouse as an animal model. Diabetes affected IL-1 beta in a tissue specific manner. For example, IL-1 beta levels (as measured by ELISA) were markedly decreased in the liver and spleen of the STZ diabetic mice. In contrast, the levels of this cytokine remained unalatered in other tissues including kidney, testis, hippocampus and pituitary. Insulin treatment restored the diabetes-related decreases in liver and spleen IL-1 beta levels. Overall, the present data suggest that the abnormalities in hepatic and splenic IL-1 beta levels may contribute, at least in part, to the immunodeficiency and increased susceptibility to infection in diabetes mellitus.