In vivo studies on age dependency of DNA repair with age in mouse skin.

Since the capacity for DNA repair relative to other cellular processes is an important parameter relevant to mutagenesis, carcinogenesis, and also aging, its assessment should preferably be carried out in intact animals. For this reason we developed an autoradiographic technique for measuring DNA repair directly in vivo. By this method unscheduled DNA synthesis (UDS) can be detected quantitatively as silver grains over epithelial cells of mouse skin after treatment with chemical carcinogens or ultraviolet (UV) irradiation. Possible age-related change in UDS response was examined by this skin technique using 2- and 18-mo-old mice. Similar dose-dependent induction of UDS was observed in mice of both ages after treatment with 4-hydroxyaminoquinoline 1-oxide. The dose-response curves for young and aged animals after UV irradiation also showed similar increases to a plateau level at low doses, but their responses to high doses were very different. In aged mice the UDS level decreased markedly with increase in dose, whereas in young mice it remained at the same plateau level. This suggests that, in aged animals, high doses of UV irradiation cause deterioration of DNA repair systems, and that aged animals cannot repair extensive UV-induced DNA damage efficiently.

Evidence of repair of DNA damage induced by 4-hydroxyaminoquinoline 1-oxide in guinea pig pancreatic slices in vitro.

In vitro exposure of guinea pig pancreatic slices to 4-hydroxyaminoquinoline 1-oxide (HAQO) resulted in increased [methyl-3H]thymidine ([3H]TdR) incorporation into DNA, both in the presence and absence of hydroxyurea (HU). Normal DNA replicative synthesis, but not DNA repair synthesis, was suppressed by HU. The increase in [3H]TdR incorporation into DNA damage induced by HAQO. Exposure of pancreatic slices to 10(-6) to 10(-5) M concentrations of HAQO did not significantly increase thymidine incorporation; however, a 15-min exposure to 10(-4) M HAQO induced a significant increase in HU-insensitive [3H]TdR incorporation into DNA. Kinetics of [3H]TdR incorporation suggests that most of the DNA repair synthesis occurs during the 2 hr following HAQO-induced DNA damage.

Changes in sterol metabolism in the skin of developing chick embryo and alterations in the presence of an anticholesterolemic agent and a chemical carcinogen.

Changes in sterol metabolism in the skin of chick embryo during its development were studied with embryonal chick skin and with the cultured skin tissues. Changes in sterol metabolism of the skin of chick embryo began to appear at day 17, as observed by the accumulation of dihydrolanosterol, and the ratio of dihydrolanostrol:cholesterol increased thereafter until hatching. A similar change in sterol metabolism was also observed with the cultured skin tissue of chick embryo, although the stages of development seem to have been delayed by 3 days. The active sterol metabolism of the cultured skin tissue was also confirmed by studies of incorporation of [2-14C]acetate into sterols. 20,25-Diazacholesterol almost completely inhibited the incorporation of [2-14C]acetate into C27 sterols, whereas a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide, inhibited the incorporation of [2-14C]acetate into lathosterol but not that into cholesterol.

A structure-carcinogenicity study of 4-nitroquinoline 1-oxides using the SIMCA method of pattern recognition.

Structure-carcinogenicity data for a series of 4-nitro- and 4-hydroxyaminoquinoline 1-oxides were analyzed using the SIMCA method of pattern recognition. Using physicochemically based substituent constants to describe each compound, a principal components model was derived for the carcinogens. This model was 82% successful in predicting the carcinogenic potential of the compounds. For the 6-substituted compounds, a significant relationship between those structural parameters associated with carcinogenic potential and ability to stimulate unscheduled DNA synthesis was observed. In addition, other problems unique to the classification of carcinogens were discussed.

Effects of ascorbate and ATP upon amino acid transport in the toad's cornea.

We have examined the effects of ascorbate upon amino acid uptake by the in vitro toad cornea. Physiologic levels of ascorbate increase the uptake of leucine by approximately 35% but have no effect upon the uptake of alanine. Uncouplers of oxidative phosphorylation do not inhibit the stimulation by ascorbic acid of leucine accumulation, indicating the increased synthesis of ATP is not the mechanism; exogenous ATP, unlike ascorbate, stimulates the uptake of both alanine and leucine. Carbon monoxide blocks the effects of ascorbate, whereas 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), which inhibits "reverse" electron transfer, enhances the accumulation of leucine. The evidence suggests that ascorbate serves as an energy source for the uptake of leucine.

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.

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.

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.

Differential effects of 4-hydroxyaminoquinoline-1-oxide on pancreatic and liver DNA synthesis in rats.

The effect of 4-hydroxyaminoquinoline-1-oxide (4-HAQO) on DNA synthesis in the pancreas and liver, target and non-target organs for 4-HAQO carcinogenesis, respectively, were compared. Pancreatic and liver DNA synthesis were simultaneously induced in rats fed a protein deficient diet containing 0.5% DL-ethionine for 18 days, and DNA synthesis in both tissues was inhibited by hydroxyurea. A single i.v. injection of 4-HAQO at a dose of 7 mg/kg body weight also inhibited DNA synthesis in both tissues within 4 h. In the pancreas the inhibition was maximum at a dose of 7 mg/kg, and DNA synthesis was less than in the pancreas of rats fed a control grain diet. This inhibition continued for the subsequent 5 days which were tested. In the liver, the degree of inhibition was less than in pancreas but the value remained higher than in rats fed control diet. The inhibition of liver DNA synthesis at a dose of 7 mg/kg completely recovered within 1 day. These results suggest that the lesions of DNA induced by 4-HAQO and its repair might be different between the pancreas and the liver. A pancreatic chemical carcinogen, 4-HAQO, might thus have the same cytotoxic effect that liver carcinogens have toward the liver resulting in failure to respond to mitotic stimuli. This might be causally related to the organotropism of 4-HAQO toward the pancreas.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. III. 4-Nitroquinoline 1-oxide and its derivatives.

Sensitivity to the chemical carcinogens 4-nitroquinoline 1-oxide (4-NQO) and 4-hydroxyaminoquinoline I-oxide (4-HAQO), during the cell cycle of synchronized HeLa S3 cells, decreases from the late S to the early G2 phases. Cells in other phases are relatively sensitive to both carcinogens. [3-H]4-NQO and [ 3-H]4-HAQO seem to be bound preferably more with cellular DNA of the mitotic phase to the middle of the S phase than with that of the late S phase in which the cells are rather insensitive to these carcinogens. However, we found no significant difference in the excision rates of these carcinogens from the DNA of HeLa S3 cells through the cell cycle. These findings indicate that the cyclic variation of 4-NQO and 4-HAQO cell survivals during the cell cycle may be due to the differences in the amounts of 4-NQO and 4-HAQO bound with cellular DNA.

Analyses of differential sensitivities of synchronized HeLa S3 cells to radiations and chemical carcinogens during the cell cycle. Par V. Radiation- and chemical carcinogen-induced mutagenesis.

8-Azaguanine(8AG)-resistant mutations induced by X-rays, ultraviolet radiation (UV) and a chemical carcinogen, 4-hydroxyaminoquinoline 1-oxide (4-HAQO) were examined during the cell cycle of synchronized HeLa S3 cells. Mutants induced by 400 R of X-rays occurred in a higher frequency in the X-ray-SENSITIVE Gl-S boundary phase than in the X-ray-resistant G1, S and early G2 phases. 8AG-resistant mutants induced by treatment with 10(-5) M 4-HAQO for 20 min appeared in a higher frequency in the early to middle S phases than in the other phases. In the case of UV, however, we found no significant difference in the induced mutation frequencies during the cell cycle, because the mutation frequencies induced by the UV doses (0-20 J/m2) used were too low for detection of the difference. These results suggest that there is a close correlation between the critical damage induced in DNA molecule(s) at the DNA-synthetic phase in the cell cycle and mutagenesis, because mitotic cells have a low mutability in spite of their high radio-sensitivity.

In vitro enzymatic recognition of DNA modified by O,O'-diacetyl or O-acetyl derivatives of the carcinogen 4-hydroxyaminoquinoline-1-oxide.

Purified DNA was modified in vitro by 3H-labelled O-acetyl or O,O'-diacetyl-4-hydroxyaminoquinoline-1-oxide (Ac4HAQO or di Ac-4HAQO). It was then subjected to the action of the single-stranded DNA specific nuclease S1 and the digested fractions were analysed. For both types of modified DNA, the release of non-modified nucleotides was faster than the release of modified nucleotides. This result is at variance with that obtained with acetoxy-acetylaminofluorene-modified DNA: in the latter case, the modified nucleotides were preferentially released. The results suggest that the S1 endonuclease can recognize different conformational changes in DNA, which depend on the carcinogen used. The enzymatic activity (or activities) present in Micrococcus luteus cell extracts released ethanol-soluble products from Ac-4HAQO modified DNA.

Experimental studies on malignant fibrous histiocytomas. II. Ultrastructure of malignant fibrous histiocytomas induced by 4-(hydroxyamino)-quinoline 1-oxide in rats.

The ultrastructures of six subcutaneous and six bone malignant fibrous histiocytomas (MFH) induced in rats by local application of the carcinogen, 4-(hydroxyamino)-quinoline 1-oxide (4-HAQO) were studied. The MFHs could be classified histologically into three subtypes: of the six subcutaneous MFHs, four were fibrous, one was giant cell, and one was myxoid; of the osseous MFHs, three were fibrous, one was giant cell, and two were myxoid. Five different types of cells were found in the MFHs: fibroblast-like cells, histiocyte-like cells, undifferentiated cells, xanthomatous cells, and multinucleated giant cells; the xanthomatous cells and multinucleated giant cells, however, were probably derived from histiocyte-like cells. Fibroblast-like cells predominated in storiform areas of the fibrous subtype; histiocyte-like cells and undifferentiated cells predominated in the giant cell subtype; intermediate cells predominated in the myxoid subtype. Acid phosphatase activity was found in lysosomes and myelin figures of the histiocyte-like cells in fibrous type MFH. The giant cell subtype of bone MFH has been transplanted serially into syngeneic rats and is now at the 17th generation. Transplantability exceeded 80%; doubling time was 3.8 to 6.1 days. Until the 3rd generation, the histology of the original tumor was retained; from the 4th generation, however, giant cells and xanthoma cells were no longer observed, and the tumor was composed mainly of undifferentiated cells. These results indicate that (a) MFH induced in the rat by 4-HAQO have an ultrastructure similar to human MFH and (b) the giant cell subtype transplanted serially is gradually transformed with a probable selection of stem cells and undifferentiated cells.

Defective repair of a class of 4NQO-induced alkali-labile DNA lesions in xeroderma pigmentosum complementation group A fibroblasts.

Normal human or xeroderma pigmentosum complementation group A (XP-A) fibroblasts were exposed to various concentrations of either 4-nitroquinoline 1-oxide (4NQO) or its 3-methyl derivative, and the rates of repair of the alkali-labile lesions induced in DNA by each agent were monitored over a period of 24 h post-treatment incubation. The data indicate that 4NQO induces at least two major classes of alkali-labile lesions into human DNA; one class disappears rapidly from the DNA of both normal and XP-A fibroblasts, while the other class undergoes repair at a relatively slow rate in normal cells, but is not removed at all in the excision-deficient cells. Methylation of 4NQO at the 3-position appears to abolish the induction of the latter class of alkali-labile lesions, whereas the rapidly removed lesions are still being induced.

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.

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.

Effect of chemical carcinogens on pancreatic DNA synthesis in vivo.

The effect of dimethylnitrosamine (DMN), ethionine, streptozotocin, N-methyl-N-nitrosourethan (MNUT), N-bis(2-hydroxypropyl)nitrosamine (DHPN), azaserine, and 4-hydorxyaminoquinoline 1-oxide (4-HAQO) on DNA synthesis after partial pancreatectomy was studied. Its results indicates that DNA synthesis of the residual pancreas occurred and reached the maximum value at 3 days and returned to the control value 12 days after the operation. DNA synthesis, which was inhibited 96.7% by hydroxyurea 3 days after the operation, indicated that semi-conservative DNA synthesis had occurred in the residual pancreas. DMN and ethionine, non-pancreatic carcinogens, did not inhibit while DHPN, MNUT, azaserine, and 4-HAQO, pancreatic non-endocrine carcinogens, inhibited DNA synthesis in the rat pancreas by 57.9, 76.4, 71.7, and 82.1%, respectively. The effect of streptozotocin, pancreatic endocrine carcinogen, on DNA synthesis was not clear from the present experiment. Further effect on the inhibition of DNA synthesis by these carcinogens was obtained by dose-response studies and its results indicated that there was a correlation between pancreatic carcinogens and the inhibition of DNA synthesis after partial pancreatectomy in rats.

Reduction of 4-nitroquinoline 1-oxide to 4-hydroxyaminoquinoline 1-oxide in lysates of cytomegalovirus-infected cells.

The rates of virus inactivation by 4-nitroquinoline 1-oxide (NQO) and 4-hydroxyaminoquinoline 1-oxide (HAQO) were compared and samples of cytomegalovirus (CMV)-infected cell lysates to which NQO had been added were examined for the presence of HAQO. These experiments demonstrated that (i) CMV inactivation by HAQO was more rapid than with NQO, (ii) virus inactivation by either NQO or HAQO failed to demonstrate a photodynamic component, and (iii) NQO-treated stocks contained HAQO, indicating reduction of NQO to HAQO. The results support the concept that metabolism of NQO to HAQO enhances the genotoxic effect of NQO.