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S G Chaney

Publications and source records attributed to S G Chaney.

At least 37 records · Page 2Linked to original sources
PubMed1997

DNA damage inducible-gene expression following platinum treatment in human ovarian carcinoma cell lines.

PURPOSE: DNA damage-inducible genes, such as gadd153, gadd45, p21 and c-jun, have previously been shown to be induced by the chemotherapeutic agent cisplatin. One of these genes, gadd153, has previously been reported to be differentially expressed in cisplatin-resistant cell lines and, therefore, to be a potential prognostic indicator for tumor response to cisplatin-based chemotherapy. It is not currently known whether such damage-inducible genes are turned on by the DNA damage itself (e.g. by the formation of Pt-DNA adducts) or by the downstream biological consequences of that damage. It is also not known whether the increased expression of these DNA-damage-inducible genes is related to immediate protective responses such as DNA repair or to more delayed responses such as cell cycle arrest or apoptosis. These experiments were initiated to characterize more fully the nature of the DNA damage-inducible response to cisplatin treatment and to determine whether any of these genes might be useful prognostic indicators of tumor response to cisplatin chemotherapy. METHODS: The dose-response and time-course for the induction of the DNA damage-inducible genes gadd153, gadd45, p21 and c-jun were examined by Northern analysis in the human ovarian carcinoma cell line 2008 and its resistant subclone C13* following treatment with platinum anticancer agents. The extent of gene expression was correlated with cytotoxicity determined by growth inhibition assay, Pt-DNA adducts determined by atomic absorption spectrometry and inhibition of DNA synthesis determined by 3H-thymidine incorporation. RESULTS: All four genes were induced maximally in both sensitive and resistant cell lines at lethal cisplatin doses (> or = ID90). Induction was maximal between 24 and 48 h following exposure to the drug for all genes except c-jun which was induced by 6 h. At 24 h following cisplatin treatment the overall levels of gadd153 were less in the resistant C13* cell line than in the parental 2008 cell line, while those of gadd45 were greater in C13* than in 2008. Maximal expression of p21 and c-jun was not significantly different in the two cell lines. The dose-response of these genes correlated with the cytotoxicity of cisplatin and the inhibition of DNA synthesis by cisplatin, rather than to the actual levels of Pt-DNA adducts. The more cytotoxic platinum analog, ormaplatin, also induced gadd153 and its induction was also based on cytotoxicity. CONCLUSION: These results suggest that the regulation of gadd153 and gadd45 expression occurs thorough separate pathways in the 2008 and C13* cell lines. The DNA damage-inducible gene response for all four damage-inducible genes tested appeared to be more directly correlated with downstream biologic effects of cisplatin damage than with actual Pt-DNA adduct levels. The time-course and dose-response for induction of these genes was more consistent with delayed responses such as apoptosis rather than more immediate responses such as DNA repair. Finally, these results strengthen previous suggestions that the expression of gadd153, and possibly other DNA damage-inducible genes, may be useful indicators of tumor response to cisplatin-based chemotherapy.

Antineoplastic Agents↗
PubMed1996

DNA repair: enzymatic mechanisms and relevance to drug response.

A number of chemotherapeutic agents, such as platinum drugs, nitrogen mustards, and chloroethylnitrosoureas, act by forming bifunctional DNA adducts. It is likely that abortive attempts to replicate and/or repair the damaged DNA cause chromosome aberrations and breakage, leading to cell death. Any substantial increase in cellular capacity to repair damaged DNA may result in resistance to chemotherapeutic agents. In this review, we examine the types of DNA adducts formed by the major classes of chemotherapeutic agents, the enzymatic pathways that play a role in the repair of those adducts, the evidence that DNA repair is enhanced in drug-resistant cell lines and tumors, and strategies for utilizing selective inhibition of DNA repair to overcome resistance.

Animals↗
PubMed1996

Increased gene specific repair of cisplatin induced interstrand crosslinks in cisplatin resistant cell lines, and studies on carrier ligand specificity.

Development of resistance to cisplatin in previously treatment-responsive malignancies is a major obstacle to successful treatment. Enhanced DNA repair as well as enhanced replicative bypass of DNA adducts have been suggested to play a role in the development of resistance to cisplatin. However, the relative contribution of these mechanisms is unknown. Second generation platinum compounds containing the 1,2-diaminocyclohexane (dach) carrier ligand have been of particular interest in the studies of resistance mechanisms since they have been effective in treatment of cells resistant to cisplatin. We have investigated the formation and repair of interstrand crosslinks (ICL) in the mouse leukemia cell line L1210/0 and its carrier ligand specific resistant derivatives L1210/DDP and L1210/DACH after treatment with ethylenediamine (en)-Pt and diaminocyclohexane (dach)-Pt compounds. ICL in the overall genome were examined using a modification of the alkaline elution assay. A Southern blot technique was employed for the study of ICL in specific regions of the genome. In the overall genome we found decreased formation of ICL with either -en or -dach carrier ligands in the two resistant cell lines without carrier ligand specificity. Some carrier ligand specificity of ICL formation was observed in the dihydrofolate reductase (DHFR) gene, but it did not correlate with the carrier ligand specificity of resistance. At the level of the overall genome there was no difference in repair of ICL between the sensitive and the two resistant cell lines. When measured in the DHFR gene, however, there was enhanced repair of ICL in the two resistant cell lines compared with the sensitive cell line. The enhanced repair at the level of the gene did not display any carrier ligand specificity.

Animals↗
PubMed1996

Cisplatin-induced alterations in the expression of the mRNAs for UV-damage recognition protein.

Enhanced DNA repair is believed to be an important mechanism of the cisplatin-resistant phenotype. UV-damage recognition protein (UV-DRP) recognizes and binds to DNA lesions and may play a role in DNA nucleotide excision repair and/or replicative bypass (which is associated with post-replication repair). Potential alternations in the expression of mRNAs for UV-DRP were analyzed in this study. Two pairs of parental and cisplatin-resistant human ovarian carcinoma cell lines were utilized. Gene expression level was assessed by northern blot hybridization. No alterations in mRNA levels for the large subunit of UV-DRP were found following cisplatin treatment, whereas mRNA levels for the small subunit of UV-DRP were induced up to 4.5-fold. The time-course and concentration-response of this induction corresponded to the previously reported increase in the UV-DRP binding activity, as measured by gel shift assay. UV-DRP binding activity in cell extracts corresponds to expression of small subunit mRNA but not to expression of large subunit mRNA. These data suggest that the small subunit may be limiting for UV-DRP activity.

Antineoplastic Agents↗
PubMed1995

HPLC and 31P NMR characterization of the reaction between antitumor platinum agents and the phosphorothioate chemoprotective agent S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721).

In prior studies, we examined the effects of the radioprotective and chemoprotective agent WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] on the in vivo biotransformation of the cisplatin [cis-diamminedichloroplatinum(II)] analog ormaplatin [(d,I)trans-1,2-diaminocyclohexanetetrachloroplatinum(IV), Pt(dach)Cl4, (formerly called tetraplatin)]. Those data suggested that a direct interaction between WR-2721 and ormaplatin and/or the corresponding Pt(II) drug, Pt(dach)Cl2, may be occurring in vivo. This would be in contrast to the generally accepted hypothesis that WR-2721 is a prodrug that must first be converted by alkaline phosphatase to a free thiol compound, WR-1065, before any appreciable reactivity would be evident. However, the major biotransformation product observed in the peritoneal fluid, plasma, and all tissues was Pt(dach)(WR-1065). We report here on further investigations into the in vitro reactivity of Pt(dach) compounds with WR-2721 and WR-1065. Separation of reaction products resulting from incubation of Pt(dach)(malonato) with either WR-2721 or WR-1065 under physiological conditions gave profiles that were indistinguishable by reverse phase HPLC and cation exchange HPLC at two different pHs. 31P NMR characterization of the dephosphorylation of WR-2721 revealed essentially no loss of inorganic phosphate for up to 24 hr when incubated in unbuffered water at 30 degrees. In contrast, when incubated with a 1:1 molar ratio of cisplatin under the same conditions, the WR-2721 signal was decreased markedly in the first 5 min, and had disappeared almost completely by 1 hr. The signal corresponding to inorganic phosphate increased in parallel to the decrease in the WR-2721 signal. No intermediate formation of a complex containing both platinum and phosphate could be detected at any time. These data suggest that the reaction between WR-2721 and platinum complexes results in rapid dephosphorylation of WR-2721, and, consequently, that the reaction products formed with either WR-2721 or WR-1065 and Pt(II) complexes are identical.

Amifostine↗
PubMed1995

Effect of the chemoprotective agent WR-2721 on disposition and biotransformations of ormaplatin in the Fischer 344 rat bearing a fibrosarcoma.

The effects of the phosphorothioate agent, WR-2721, have been investigated with respect to the biotransformations of ormaplatin in the Fischer 344 rat bearing a transplanted fibrosarcoma. A number of different paradigms of dosing route and schedule for the administration of the two agents have been investigated. In the first group of experiments, WR-2721 (200 mg/kg, i.p.) was administered 30 min before ormaplatin (12.5 mg/kg, i.p.), and then peritoneal fluid, plasma, and tissues were harvested at 30 min after the ormaplatin administration. Our results suggest that a significant interaction between WR-2721 and ormaplatin is occurring in the peritoneal cavity. The interaction was evident in terms of both effects on distribution and disposition of total platinum and in alterations of the profiles of biotransformation products formed in the various tissues and fluids. Plasma protein binding of ormaplatin was decreased by 50% in the presence of WR-2721. Total platinum in the spleen was decreased by 66% and in the liver by 50%. There were no trends among the findings that would indicate any selectivity between tumor and nontumor tissue with respect to the effects of WR-2721 on the parameters measured. Subsequent investigations examined the effects of dosing the WR-2721 by the i.v. route while continuing with the i.p. administration of the ormaplatin. WR-2721 was administered either 30 or 5 min before the ormaplatin, and the plasma and tissues were harvested at 15, 30, or 60 min after ormaplatin administration. The reverse-phase HPLC peak, which behaved chromatographically as a Pt(dach)(WR-1065) standard, was less prominent after the i.v. administration of WR-2721 than it was after i.p. administration under any of the paradigms tested. There was again no evidence for selectivity between tumor and nontumor tissue in the findings from any of the paradigms. It is concluded that if WR-2721 is capable of selectively protecting nontumor tissue from the toxicities of platinum-based chemotherapy, it is doing so by some mechanism other than its selective uptake into normal tissue and subsequent nonspecific inactivation of any reactive cytosolic platinum species formed. Other possible mechanisms are briefly discussed.

Amifostine↗
PubMed1995

Induction of UV-damage recognition protein by cisplatin treatment.

The biological functions of DNA damage recognition proteins are not well understood. Using the band shift assay, we detected in nuclear extracts from human carcinoma cell lines damage recognition protein which bound selectively to UV-damaged double-stranded DNA. No consistent correlation was found between steady-state levels of the UV-damage recognition protein and either cisplatin cytotoxicity or DNA repair activity. However, cisplatin treatment caused accumulation of the UV-damage recognition protein. The cisplatin-responsive induction of UV-damage recognition protein in the nucleus was higher in cisplatin-resistant cell lines than in their parental counterparts. These results imply that the level of inducibility in response to treatment, but not the constitutive binding activity, of UV-damage recognition protein correlates with cisplatin resistance. Inhibition of UV-damage recognition protein expression by actinomycin and cycloheximide suggests that induction of UV-damage recognition protein requires de novo RNA and protein synthesis, rather than post-translational modification of pre-existing protein. The increased level of UV-damage recognition protein after cisplatin treatment could be a direct response to adduct formation, since it correlated with the number of Pt-DNA adducts. However, it could also be a secondary effect of DNA replication inhibition following DNA damage, since inhibition of DNA synthesis by aphidicolin and hydroxyurea caused the same induction of UV-damage recognition protein. Inducibility of UV-damage recognition protein binding activity by Pt drug treatment suggests involvement of this protein in drug resistance, although a direct link between its function and DNA repair or damage tolerance has not been demonstrated.

Cell Cycle↗
PubMed1995

Organ-specific biotransformation of ormaplatin in the Fischer 344 rat.

We examined the intracellular biotransformation products of ormaplatin [(d,l-trans)1,2-diaminocyclohexanetetrachloroplatinum(IV)] (formerly called tetraplatin) in liver, kidney, spleen, small intestine, and plasma of the adult male Fischer 344 rat. Previous studies have established that the rank order of ormaplatin toxicity in Fischer 344 rats is spleen approximately gastrointestinal tract > kidney >> liver. Animals were given tritium-labelled drug i.v. at 12.5 mg/kg, and tissues were harvested 30 min later. The kidney was found to concentrate total and cytosolic platinum to a greater extent than any of the other tissues. The absolute amount of cytosolic platinum, in micrograms per gram tissue, that was irreversibly bound to protein and/or other macromolecules was also greatest in the kidney. However, when the amount bound was expressed as a percentage of the total cytosolic platinum, the kidney was significantly lower than any other tissue. Of the various low molecular mass platinum biotransformation species characterized, by far the most abundant were complexes of platinum with the sulfur-containing molecules cysteine, methionine, and glutathione (GSH). There was more of the methionine complex in the blood plasma than in any of the tissues except for the spleen. No significant differences among the tissues were detected for the dichloro, cysteine, methionine, or the GSH complexes. The tritium-labelled diaminocyclohexane (DACH) carrier ligand appeared to remain stably bound to the platinum while in the plasma, as there was less free DACH ligand detected in plasma ultrafiltrate than in any tissue ultrafiltrate. Among the tissues, the free DACH levels were in the range of 20% of the radioactivity recovered from the HPLC column and were not significantly different. Consequently, neither biodistribution nor tissue-specific biotransformation of ormaplatin provides a ready explanation for the tissue specificity of ormaplatin toxicity in Fischer 344 rats. However, in kidney there was much less of the reactive PtCl2(DACH) species than has previously been reported for the corresponding Pt(NH3)2Cl2 species in cisplatin-treated rats. Thus, these data suggest a possible explanation for differences in nephrotoxicity induced by cisplatin versus that by ormaplatin.

Analysis of Variance↗
PubMed1995

Possible correlation between ormaplatin biotransformations and neurotoxicity.

Clinical development of ormaplatin has been delayed because of neurotoxicity that was not predictable on the basis of patient characteristics, total cumulative dose, or plasma pharmacokinetics. We report a detailed comparison of the plasma biotransformations of ormaplatin in two patients at the 123 mg/m2-dose administered as a 1-h infusion every 4 weeks. One of these patients developed neurotoxicity after 2 cycles (total cumulative dose = 246 mg/kg), while the other patient showed no symptoms of neurotoxicity through 2 cycles. The maximum plasma concentration and area under the curve for ultrafilterable platinum were greater for the patient that did not develop neurotoxicity. However, both maximum plasma concentration and area under the curve were greater for dichloro(d,l-trans)1,2-diaminocyclohexanedichloroplatinum(II), the major active biotransformation product of ormaplatin, in the patient that developed neurotoxicity. In addition, analysis of plasma biotransformation products suggested that the initial plasma concentrations of ormaplatin were also greater in the patient that developed neurotoxicity. These data suggest that analysis of individual plasma biotransformation products may be useful in predicting toxicity of platinum anticancer agents and should be included in future phase I studies.

Adult↗
PubMed1994

Enhanced replicative bypass of platinum-DNA adducts in cisplatin-resistant human ovarian carcinoma cell lines.

We have examined the relationship between cis-diamminedichloroplatinum(II) (cisplatin) resistance and replicative bypass in the human ovarian carcinoma cell lines 2008, A2780, and their respective cisplatin-resistant derivatives C13* and A2780/DDP. Replicative bypass is defined as the ability of a replication complex to proceed past a DNA adduct known to block or stall the complex during synthesis. Previous studies in our laboratory have shown a 3-4-fold increase in the replicative bypass of platinum-DNA adducts in platinum-resistant murine leukemia cell lines [G. R. Gibbons et al, Carcinogenesis (Lond.), 12: 2253-2257, 1991]. To test for this effect in the human lines, we used a steady-state replication assay which measures the inhibition of DNA chain elongation (based on the incorporation of [3H]thymidine into nascent DNA strands) as a function of the number of platinum-DNA adducts present on the DNA following cisplatin treatment. With this technique we demonstrated a 4.5-fold increase in the replicative bypass ability of the C13* line compared to the 2008 line and a 2.3-fold increase in the bypass ability of the A2780/DDP line compared to the A2780 line. To confirm these results, we performed a pulse-chase replication assay on the 2008 and C13* lines. This assay differs from the first in that DNA chain elongation is measured in a time-dependent manner. With the pulse-chase assay we observed a 4.8-fold increase in the replicative bypass ability of the C13* line compared to the 2008 line. We then examined the specificity of this enhanced bypass by repeating the steady-state assay with the 2008 and C13* lines using as damaging agents 1,2-diaminocyclohexanedichloroplatinum(II), UV radiation (producing pyrimidine dimers), and benzo(a)pyrene-7,8-diol-9,10-epoxide. In both cell lines, 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adducts caused a greater inhibition of DNA chain elongation than cisplatin-DNA adducts. The level of enhanced bypass of 1,2-diaminocyclohexanedichloroplatinum(II)-DNA adducts in the resistant line was 2.1-fold (approximately 2-fold less than the level of enhanced bypass observed with cisplatin-DNA adducts). There was no evidence of enhanced bypass in the resistant line when cells were treated with UV light or benzo(a)pyrene-7,8-diol-9,10-epoxide. These results indicate that the bypass response in the C13* line has some degree of specificity for cisplatin adducts. The specificity of bypass in these cell lines coincided well with the specificity of resistance to each agent.(ABSTRACT TRUNCATED AT 400 WORDS)

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1994

Pharmacokinetic and biotransformation studies of ormaplatin in conjunction with a phase I clinical trial.

Ormaplatin is a second-generation platinum (Pt) analogue with in vitro activity against some cisplatin-resistant malignant cell lines. We have evaluated the pharmacokinetics and biotransformations of ormaplatin during a phase I trial in which ormaplatin was administered by daily 30-min infusions on 5 consecutive days every 28 days. Sixteen patients received 25 courses at doses ranging from 5.0 to 11.6 mg/m2 per day. Pharmacokinetic parameters determined for ultrafilterable Pt measured by atomic absorption spectrophotometry revealed a short half-life (t1/2 16 min), moderate volume of distribution (Vd 12 l/m2), and relatively fast systemic clearance (Cls 544 ml/min per m2). Cls and percentage of drug unbound decreased during the 5-day administration period. Average systemic exposure increased with dose; however, inter-individual variability in Cls produced overlap in systemic exposure between the dose levels. The major active biotransformation product [PtCl2(dach)] was evaluated at the highest dose level by HPLC. This product decayed monoexponentially with a mean t1/2 of 13 min and a higher degree of pharmacokinetic variability than that of ultrafilterable Pt at this dose. No unreacted ormaplatin was detected; however, several inactive biotransformation products persisted for at least 120 min. Approximately 32% of the dose was excreted in the urine during the first day, one-third of this during the initial 1.5 h. The human pharmacokinetic characteristics of ormaplatin resemble those of cisplatin; however, additional study will be required to discern which analyte of ormaplatin correlates best with clinical effects.

Adult↗
PubMed1993

Role of carrier ligand in platinum resistance of human carcinoma cell lines.

We have examined the effects of the cis-diammine and 1,2-diaminocyclohexane (dach) carrier ligands on cytotoxicity, platinum accumulation and efflux, platinum incorporation into DNA, cytotoxicity of Pt-DNA adducts, and repair of Pt-DNA adducts in the human ovarian carcinoma A2780 cell line, the human colon carcinoma HCT8 cell line, and their cis-diamminedichloroplatinum(II) (cisplatin)-resistant derivatives, A2780/DDP and HCT8/DDP. The A2780/DDP cell line was 7.7-fold resistant to cisplatin, and the HCT8/DDP cell line was 1.6-fold resistant to cisplatin compared to their parental cell lines. Both were considered as examples of acquired cisplatin resistance. The HCT8/S cell line was 4.6-fold resistant to cisplatin compared with the A2780/S cell line and was considered an example of intrinsic resistance. Decreased accumulation of cisplatin made a significant contribution to acquired cisplatin resistance in the A2780/DDP cell line, probably contributed to intrinsic resistance in the HCT8/S cell line, but made little or no contribution to acquired resistance in the HCT8/DDP cell line. Decreased cytotoxicity of Pt-DNA adducts made a major contribution to both acquired and intrinsic cisplatin resistance in all three cell lines. Increased repair activity made a significant contribution to the decreased cytotoxicity of Pt-DNA adducts in the HCT8/S cell line, a weak contribution in the A2780/DDP cell line, and no contribution in the HCT8/DDP cell line. Glutathione levels were elevated in all the cell lines with acquired and intrinsic resistance, but the increased glutathione levels were not associated with decreased incorporation of platinum into DNA. These data suggest that both decreased accumulation and increased repair contribute to cisplatin resistance to different degrees in these human carcinoma cell lines. In addition, mechanism(s) other than repair may contribute to the decreased cytotoxicity of cis-diammine-Pt-DNA adducts. Of the cells with acquired cisplatin resistance, the HCT8/DDP cell line showed no resistance to tetrachloro(trans-DL)1,2-diaminocyclohexaneplatinum(IV) (ormaplatin, formerly known as tetraplatin), while the A2780/DDP cell line was just as resistant to ormaplatin as to cisplatin. The intrinsically cisplatin-resistant HCT8/S cell line showed only partial cross-resistance to ormaplatin. The effects of the dach carrier ligand on both acquired and intrinsic resistance in these cell lines appeared to occur primarily at the level of cytotoxicity of dach-Pt adducts, but the differences in the cytotoxicity of cis-diammine-Pt and dach-Pt adducts could not be explained by differences in repair of those adducts.(ABSTRACT TRUNCATED AT 400 WORDS)

Antineoplastic Agents↗
PubMed1992

Limitations of the in vitro repair synthesis assay for probing the role of DNA repair in platinum resistance.

Several studies have implicated enhanced DNA repair in acquired platinum resistance. To better understand the mechanism of increased repair we have employed an in vitro assay using cell-free extracts from platinum sensitive and resistant murine and human cell lines. Since the platinum resistant murine cell lines used in our previous studies had shown increased repair of diaminocyclohexane(dach)-Pt-DNA adducts while one of the resistant human cell lines did not, we have measured in vitro repair synthesis on DNA damaged by (d,l)-trans-1,2-diaminocyclohexanedichloroplatinum(II) (PtCl2(dach)). The results of this assay were strongly dependent on the method used to calculate repair synthesis activity and appeared to disagree with previous estimates of repair activity in these cell lines. By one method of calculation the in vitro repair synthesis assay underestimated the ratio of repair activities in the resistant versus the sensitive murine cell lines, while by the other method the in vitro assay overestimated the ratio of repair activities in the resistant versus the sensitive human cell lines.

Animals↗
PubMed1991

An unexpected biotransformation pathway for tetrachloro-(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in the L1210 cell line.

Tetrachloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) has been considered a prodrug which would be converted rapidly to dichloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)] under physiological conditions. However, the biotransformations of tetraplatin have not been studied in detail. We have followed the intracellular biotransformations of tetraplatin and PtCl2(dach) in the L1210 cell line by a two-step high performance liquid chromatography separation procedure described previously (Mauldin et al., Cancer Res., 48: 5136-5144, 1988). At early times the intracellular biotransformation pathways appeared to be very different in tetraplatin- and PtCl2(dach)-treated cells. The tetraplatin present in the medium initially was taken up preferentially by the L1210 cells. However, no intracellular tetraplatin and very little intracellular PtCl2(dach) were found in the tetraplatin-treated cells. Instead, two previously unidentified biotransformation products predominated at early times. The same biotransformation products were present in cells incubated in Hank's balanced salt solution, so they most likely did not arise from extracellular reactions. The unidentified biotransformation products present in tetraplatin-treated cells at early times appeared to be at the platinum(II) level of oxidation. Model reactions suggested that these compounds could have been formed by platinum(II)-assisted platinum(IV) substitution reactions, followed by reduction of the platinum(IV) complex to the platinum(II) level. Thus, there appear to exist unique features of tetraplatin metabolism which are observed only when tetraplatin is taken up directly by the cell without prior reduction. These reaction products did not react with DNA and presumably represent an inactivation pathway.

Animals↗
PubMed1991

Sequence effect on incision by (A)BC excinuclease of 4NQO adducts and UV photoproducts.

Nucleotide excision repair in Escherichia coli is initiated by (A)BC excinuclease, an enzyme which incises DNA on both sides of bulky adducts and removes the damaged nucleotide as a 12-13 base long oligomer. The incision pattern of the enzyme was examined using DNA modified by 4-nitroquinoline 1-oxide (4NQO) and UV light. Similar to the cleavage pattern of UV photoproducts and other bulky adducts, the enzyme incises the 8th phosphodiester bond 5' and 5th phosphodiester bond 3' to the 4NQO-modifed base, primarily guanine. The extent of DNA damage by these agents was determined using techniques which quantitatively cleave the DNA or stop at the site of the adduct. By comparison of the intensity of gel bands created by (A)BC excinuclease and the specific cleavage at the damaged site, the efficiency of (A)BC excinuclease incision at 13 different 4NQO-induced adducts and 13 different photoproducts was determined by densitometric scanning. In general, incisions made at 4NQO-induced adducts are proportional to the extent of damage, though the efficiency of cutting throughout the sequence tested varies from 25 to 75%. Incisions made at pyrimidine dimers are less efficient than at 4NQO-adducts, ranging from 13 to 65% incision relative to modification, though most are around 50%. The two (6-4) photoproducts within the region tested are incised more efficiently than any pyrimidine dimer.

4-Nitroquinoline-1-oxide↗
PubMed1991

In vivo and in vitro effects of helenalin on mouse hepatic microsomal cytochrome P450.

Helenalin, a natural plant product with significant antitumor activities, decreased male BDF1 mouse hepatic microsomal cytochrome P450 contents in vivo and in vitro. A single i.p. dose of 25 mg helenalin/kg body weight significantly (P less than 0.05) decreased microsomal cytochrome P450 contents and inhibited cytochrome P450-dependent mixed-function oxidase activities within 1-2 hr post-exposure. Helenalin (1.0 mM) decreased microsomal cytochrome P450 contents in vitro by 11% in the absence of NADPH and by 32% in the presence of NADPH. These in vitro and in vivo decreases in cytochrome P450 were accompanied by comparable decreases in total microsomal heme contents. Helenalin (1.0 mM) increased mouse hepatic microsomal oxygen consumption and NADPH utilization by 3.2 and 5.4 nmol/min/mg protein respectively. Helenalin (1.0 mM) significantly (P less than 0.05) increased microsomal lipid peroxidation in vitro, and this helenalin-induced increase in lipid peroxidation was inhibited completely by the addition of 0.05 mM EDTA. However, microsomal cytochrome P450 contents were equally affected by helenalin in the presence or absence of EDTA, suggesting that lipid peroxidation did not contribute to the helenalin-induced decrease in cytochrome P450. The addition of 0.05 mM hemin to microsomes treated in vitro with 1.0 mM helenalin resulted in a 58% recovery of cytochrome P450 contents. This ability of hemin to reconstitute cytochrome P450 in helenalin-treated microsomes suggests that helenalin produced a selective loss of heme from the cytochrome P450 holoprotein, and that the resulting cytochrome P450 apoprotein remained intact after helenalin treatment. The increased loss of microsomal cytochrome P450 produced by helenalin in the presence of NADPH suggests that a helenalin metabolite may be responsible for heme loss and the in vitro destruction of cytochrome P450.

Animals↗
PubMed1991

Effects of diethyldithiocarbamate (DDTC) on the plasma biotransformations of tetrachloro(d,l-trans)-1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in Fischer 344 rats.

We have studied the effects of diethyldithiocarbamate (DDTC) on the biotransformations of toxic doses of tetrachloro (d,l-trans)1,2-diaminocyclohexaneplatinum(IV) (tetraplatin) in Fischer 344 rats. In animals not treated with DDTC, tetraplatin was rapidly converted to dichloro(d,l-trans)1,2-diaminocyclohexaneplatinum(II) [PtCl2(dach)]. Subsequent biotransformations included the transient formation of the (d,l-trans)1,2-diaminocyclohexane-aquachloroplatinum(II) [Pt(H2O)(Cl)(dach)]+ complex, followed by formation of the platinum (Pt)-methionine and either Pt-cysteine or Pt-ornithine complexes. Significant amounts of free (d,l-trans) 1,2-diaminocyclohexane (dach) were observed in plasma as a result of intracellular trans-labilization reactions. DDTC caused a marked decrease in both total and protein-bound platinum in the circulation. A significant increase in the plasma concentration of free dach was also observed as a result of formation of the Pt(DDTC)2 complex. Some of the free dach could have arisen from intracellular reactions with DDTC, but the displacement of platinum from plasma proteins was more than sufficient to account for the increase in free dach in the circulation. DDTC treatment also decreased plasma concentrations of tetraplatin, PtCl2(dach), [Pt(H2O)(Cl) (dach)]+, the Pt-methionine complex, and one unidentified biotransformation product, but had no effect on the Pt-cysteine (or Pt-ornithine) complex. These effects of DDTC on protein-bound platinum and low-molecular-weight biotransformation products in plasma may contribute to the decrease in tetraplatin toxicity seen in DDTC-treated rats.

Animals↗