Differential expression of cytokines in rat osteosarcoma and malignant fibrous histiocytoma cell lines induced by 4-(hydroxyamino)quinoline-1-oxide.

Cytokines are considered to play an important role in tumor pathogenesis and progression, and recent studies have demonstrated that a variety of forms, including interleukins (ILs) and transforming growth factor-beta(s) (TGF-beta(s)), may regulate tumors. In the present study, the expression of TGF-beta isoforms and ILs was investigated in cell lines from a rat osteosarcoma and a malignant fibrous histiocytoma (MFH), both established from transplantable tumors induced by 4-(hydroxyamino) quinoline 1-oxide (4-HAQO) in syngeneic F344 male rats. The results of a multiprobe RNase protection assay showed TGF-beta1 expression to be remarkably elevated, with no TGF-beta2 and beta3 detectable in MFH cells, while TGF-beta1 and -beta2 were found to be moderately and TGF-beta3 weakly expressed in osteosarcoma lines. All cell lines of osteosarcomas and MFHs expressed macrophage migration inhibitory factor at similar levels. In contrast to the lack of ILs in the MFH cells, moderate IL-6 and very weak IL-1beta expression was detected in the osteosarcoma cells. These results suggest that variation in expression pattern of these cytokines in osteosarcomas and MFHs might be involved in differences in histological appearance and biological behavior, including metastatic ability, between these two mesenchyme-derived tumor types.

Selective 8-hydroxyguanine formation in pancreatic DNA due to a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide in rats.

8-Hydroxyguanine (8-OHG) formation, a possible initiating event, was determined in pancreatic and liver DNA and compared with the genesis of acinar cell and hepatocyte necrosis in male Wistar rats given a single intravenous administration of 4-hydroxyaminoquinoline 1-oxide (4-HAQO). At the non-necrotic but tumorigenic dose of 7.0 mg/kg body weight, 8-OHG was selectively generated in pancreatic DNA, in the absence of acinar cell necrosis, at the 6 and 24 h time points and repaired by the 48 h time point. When rats were exposed to 4-HAQO at a necrotic dose of 14.0 mg/kg body weight, 8-OHG was also selectively formed in pancreatic DNA with the same time-dependence of generation and repair, while acinar cell necrosis became evident at the 24 h time point and progressed thereafter. Whereas no hepatocyte necrosis was detected in any rats, 8-OHG values for liver DNA merely expressed slight increases only at the 24 and 48 h time points in rats given 14.0 mg/kg body weight of 4-HAQO. The present data suggest that formation of oxidative DNA damage, assayed by 8-OHG, in pancreatic DNA is independent from toxicity and may be involved, along with quinoline adducts, in mutational events underlying 4-HAQO-induced rat acinar cell carcinogenesis.

Identification of N4-(guanosin-7-yl)-4-aminoquinoline 1-oxide and its possible role in guanine C8 adduction.

A novel base adduct of the carcinogen, 4-nitroquinoline 1-oxide, N4-(guanin-7-yl)-4-amino-quinoline 1-oxide was identified from RNA, which was bioactivated 4-hydroxyaminoquinoline 1-oxide. In addition of base adducts, we uncovered the formation of 8-hydroxyguanine residue (8-OH-G) in DNA and RNA after treatment of 4NQO, in vivo and in vitro. A conceivable mechanism of formation of 8-OH-G and guanine C8-substituted quinoline adducts is proposed.

The FLP protein contacts both major and minor grooves of its recognition target sequence.

The FLP protein of the 2 microns plasmid of Saccharomyces cerevisiae promotes conservative site-specific recombination between DNA sequences that contain the FLP recognition target (FRT). FLP binds to each of the three 13 base pair symmetry elements in the FRT site in a site-specific manner. We have probed both major and minor groove contacts of FLP using dimethyl sulphate, monoacetyl-4-hydroxyaminoquinoline 1-oxide and potassium permanganate and find that the protein displays extensive interactions with residues of both the major and minor grooves of 10 base pairs of each symmetry element. We find no evidence that the FRT site assumes a single-stranded conformation upon FLP binding.

Cytomegalovirus-enhanced induction of chromosome aberrations in human peripheral blood lymphocytes treated with potent genotoxic agents.

Human cytomegalovirus (HCMV) has been shown to increase the frequency of chromosome aberrations, primarily chromatid-type, in human peripheral blood lymphocytes (PBLs). Because HCMV persists in most humans, pathologically activates cells, and may perturb the cell cycle, we investigated the possibility that HCMV-infected cells have a modified sensitivity to chromosome damage induced by genotoxic chemicals. Uninfected PBLs exposed to bleomycin (3 to 100 micrograms/ml) demonstrated a linear increase in the frequency of chromosome aberrations. HCMV infection of PBLs at an intensity that did not cause detectable damage followed by exposure to the same concentrations of bleomycin resulted in a significant enhancement (p less than 0.01) in the frequency of chromosome aberrations relative to the effect of bleomycin alone. A more than additive enhancement of the frequency of chromosome aberrations was also noted in HCMV-infected PBLs exposed to 4-hydroxyaminoquinoline-1-oxide (4-HAQO; 0.1 to 0.3 micrograms/ml) relative to uninfected cells treated with 4-HAQO alone. No increase in the percentage of aberrant cells or the frequency of chromosome aberrations was observed in HCMV-infected cells treated with 4-nitroquinoline-1-oxide (4-NQO) relative to similarly treated uninfected PBLs. These results suggest that HCMV can potentiate the induction of chromosome aberrations in human PBLs caused by potent DNA damaging agents.

Species and organ differences in DNA adduct formation and repair after treatment with 4-hydroxyaminoquinoline 1-oxide.

4-Hydroxyaminoquinoline 1-oxide (4HAQO) demonstrates obvious organotropic and species specificity in its carcinogenesis and the present investigation concerns 4HAQO DNA adduct formation and repair as studied in various organs of four animal species (rats, mice, guinea pigs and hamsters). Three hours after an iv injection of 10 mg per kg body weight of tritium-labeled 4HAQO, the major organs were removed and used for assessment of label incorporation in the DNA. The results showed that the DNA binding levels generally correlated well with the reported species and organ specificity of 4HAQO tumorigenesis. For example, rats showed highest DNA binding in the pancreas and kidney, major target organs. The levels of DNA binding in the liver were invariably low in all 4 animal species, in agreement with the lack of hepatocarcinogenicity associated with 4HAQO exposure. A clear relationship between DNA adduct formation and carcinogen dose was also found after treatment of mice with 4HAQO at doses of 1, 5, 10 and 20 mg per kg body weight in all tissues (pancreas, kidney and lung) except for the liver. Comparison of DNA repair processes in rats, a highly susceptible species, and hamsters, a resistant species in terms of 4HAQO carcinogenicity, revealed highest formation and slowest removal of adducts in the target organs of the rat. In the hamster organs and the rat lung and liver, DNA adduct formation was generally low and in the case of elevation in the initial phase, quickly removed.

Formation of 8-hydroxyguanine residues in DNA treated with 4-hydroxyaminoquinoline 1-oxide and its related compounds in the presence of seryl-AMP.

The mechanism whereby treatment of DNA with 4-hydroxyaminoquinoline 1-oxide (4HAQO) in the presence of seryl-AMP leads to the formation of 8-hydroxyguanine (8-OH-Gua) residues in DNA (Kohda et al., this journal, 139, 626, 1986) has been studied. In the survey of other N-arylhydroxylamines, only 4HAQO analogues which could bind to DNA produced 8-OH-Gua. The amount of 8-OH-Gua varied depending on the structure of 4HAQO analogues and that of DNA. The formation of 8-OH-Gua was not inhibited by active oxygen scavengers. Possible mechanisms are discussed.

Altered drug metabolizing potential of acinar cell lesions induced in rat pancreas by hydroxyaminoquinoline 1-oxide.

Foci of atypical acinar cells observed in male rats 1 year after a single injection of hydroxyaminoquinoline 1-oxide (HAQO) were assessed immunohistochemically for altered expression of a number of enzyme forms considered to play important roles in drug metabolism. The pancreatic lesions, classified as of either basophilic or eosinophilic type on histological appearance, demonstrated distinctive patterns of altered enzyme phenotype. On the one hand, the basophilic foci composed of enlarged cells/nuclei with very prominent nucleoli were characterized by increase in GST-P, G6PD and P450 PB1 and MC2 forms. The eosinophilic type, in contrast, comprised smaller cells demonstrating elevated P450 MC1 and PB1 but not MC2, normal G6PD and strong GST-P binding limited only to a proportion of the nuclei. Both shared decreased GGT and almost total lack of GST-B positive connective tissue and ductular elements. Apparent islet cell lesions and normal islet tissue were characterized by a distinct enzyme phenotype strongly positive for all P450 species investigated. The results indicate that HAQO-induced putative preneoplastic pancreatic lesions, like equivalent carcinogen associated with focal populations in liver, kidney and ductular pancreas, demonstrate a non-random altered expression of specific drug metabolizing enzyme species.

Ultrastructural changes in pancreatic acinar cells of rats after administration of 4-hydroxyaminoquinoline-1-oxide.

Ultrastructural changes in the exocrine pancreas 24, 48, 72 and 168 hours after a single intravenous injection of 4-hydroxyaminoquinoline-1-oxide (4-HAQO), at a dose of 14 mg.per kg., were observed in male rats. At 24 hours after administration, multiple focal degenerative lesion-like large vacuoles in the acini, and a decrease in zymogen granules with dilation of the rough endoplasmic reticulum cisternae in the acinar cells were marked. At 48 hours, acinar cell degeneration and necrosis progressively increased. The nucleus, especially, appeared to be disorganized and lysosome-like bodies with various sizes were frequently observed. At 72 hours, acinar cell degeneration persisted in the acini, and the interstitial space with infiltration of inflammatory cells appeared edematous. In addition, ductular-like cells, which resembled intercalated duct cells, possessing a light cytoplasm with occasional mitoses were observed around the duct lumen. At 168 hours, the exocrine pancreas was occupied with proliferated ductular-like cells. Furthermore, acinar cells and acini regenerated to the normal pattern were sometimes found. Thus, the exocrine pancreas degenerated progressively up to 48 hours after administration of 4-HAQO, gradually came to be repaired by degrees from 72 hours and then partly appeared to be regenerated at 168 hours. It is suggested that the ductular-like cell might be the precursor of the acinar cell in the regenerating process after injection of 4-HAQO.

Formation of 8-hydroxyguanine residues in cellular DNA exposed to the carcinogen 4-nitroquinoline 1-oxide.

8-Hydroxyguanine (8-OH-Gua) residues were formed in DNA of Ehrlich ascites cells exposed to the carcinogen 4-nitroquinoline 1-oxide. Formation of 8-OH-Gua was confirmed by chemical treatment of calf thymus DNA with the proximate metabolite of this carcinogen, 4-hydroxyaminoquinoline 1-oxide, together with seryl-adenosine monophosphate. The ratio of the rates of formations of 8-OH-Gua and the quinoline-bound adducts was about 0.2-0.3. A conceivable mechanism of formation of 8-OH-Gua is proposed.