Most hepatoma cell lines lack proper expression and induction of cytochrome P450 (CYP) enzymes and this deficiency hampers their use as in vitro models for drug and xenobiotic metabolism. According to previous studies, the poor expression of CYP enzymes may be due to decreases in CYP gene transcription. Two nuclear receptors (NRs), the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), are known to regulate many genes involved in xenobiotic metabolism and disposition. Here, we studied the expression of different CYP, NR and NR co-regulator genes in hepatoma cell lines. Next, we created “chimeric NR” constructs by cloning the strong activation domain from the p65 subunit of transcription factor NF-κB and appending it to either N- or C-termini of the human CAR or PXR. We established that these chimeric NRs displayed enhanced trans-activation potential as compared to the unmodified NRs, and showed that transient transfection of a single chimeric NR increased the expression of several CYPs simultaneously. Finally, stable cell lines expressing a chimeric NR had elevated levels of CYP3A4, CYP2B6 and CYP2C9 mRNAs and CYP3A4-mediated metabolism when compared to the wild-type hepatoma cells. These findings establish a proof of principle how improved metabolic cell models could be designed.

The Comet assay (microgel electrophoresis) was used to study DNA damage in Raji cells, a B-lymphoblastoid cell line, after treatment with different doses of neutrons (0.5 to 16 Gy) or gamma rays (1.4 to 44.8 Gy). A better growth recovery was observed in cells after gamma-ray treatments compared with neutron treatments. The relative biological effectiveness (RBE) of neutron in cell killing was determined to be 2.5. Initially, the number of damaged cells per unit dose was approximately the same after neutron and gamma-ray irradiation. One hour after treatment, however, the number of normal cells per unit dose was much lower for neutrons than for gamma rays, suggesting a more efficient initial repair for gamma rays. Twenty-four hours after treatment, the numbers of damaged cells per unit dose of neutrons or gamma rays were again at comparable level. Cell cycle kinetic studies showed a strong G2/M arrest at equivalent unit dose (neutrons up to 8 Gy; gamma rays up to 5.6 Gy), suggesting a period in cell cycle for DNA repair. However, only cells treated with low doses (up to 2 Gy) seemed to be capable of returning into normal cell cycle within 4 days. For the highest dose of neutrons, decline in the number of normal cells seen at already 3 days after treatment was deeper compared with equivalent unit doses of gamma rays. Our present results support different mechanisms of action by these two irradiations and suggest the generation of locally multiply damaged sites (LMDS) for high linear energy transfer (LET) radiation which are known to be repaired at lower efficiency.

The p53 gene was examined for point mutations in archived, alpha-radiation-associated lung and liver cancers. Lung tumors of 50 uranium miners in Germany were screened by restriction fragment length analysis for the putative hotspot mutation at codon 249 (Arg-->Met) previously detected in a significant fraction of miners from the Colorado Plateau, USA. This mutation has been proposed as a marker of radon exposure. None of the tumors we examined harbored the hotspot mutation. Five of the 50 tumors, however, did indeed harbor exon 7 mutations, as determined by subsequent mutation analysis of exon 7. These mutations were dispersed among various codons and may be attributable to heavy tobacco smoking in this cohort. In support of this interpretation, we found no mutations in exons 5-8 of the p53 gene in 13 iatrogenic liver cancers induced by injection of Thorotrast, an alpha-emitting radiocontrast agent. We propose that if the p53 tumor suppressor gene is a target for the carcinogenic action of alpha-particle radiation, loss of suppressor function may occur preferentially by mechanisms such as intrachromosomal deletions, rather than by base substitution mutations.

The application of a 32P-postlabeling assay for 7-methylguanines in DNA was studied either by labeling the imidazole ring-opened dinucleotide derivatives or by using strong-anion-exchange column chromatography for the adduct enrichment from normal nucleotides. Data showed that 7-methylguanines can be efficiently labeled as dinucleotides when in vitro methylated DNA was first imidazole ring-opened and then digested to the dinucleotide level with deoxyribonuclease I, snake venom phosphodiesterase, and prostatic acid phosphatase. When using ion exchange chromatography for the adduct enrichment, DNA was digested with micrococcal nuclease and spleen phosphodiesterase. Anion exchange chromatography was applied for 7-methylguanine measurements in white blood cell DNA of healthy nonsmokers (n = 17) and patients (n = 4) treated with the methylating drugs procarbazine and decarbazine. We found that the mean level of 7-methylguanine residues in nonsmokers was 2.5 per 10(7) nucleotides. The corresponding level in the patient samples immediately after the drug treatment was 57 per 10(7) nucleotides.

The effect of smoking was investigated on the formation of 7-methylguanines in human peripheral white blood cells. DNA was isolated from total white blood cells, granulocytes and lymphocytes from 10 smokers and 10 non-smokers. 32P-Postlabeling was performed by using anion-exchange chromatography enrichment of adducts. In smokers the mean DNA adduct levels were 6.9, 4.7 and 23.6 7-methylguanine residues/10(7) nucleotides in total white blood cells, granulocytes and lymphocytes respectively. The corresponding values in non-smokers were 3.4, 2.8 and 13.5 adducts/10(7) nucleotides. The mean adduct level was significantly higher in lymphocytes than in total white blood cells or granulocytes both in smokers and in non-smokers. The mean adduct levels differed significantly between smokers and non-smokers.

A 32P-postlabelling method was developed to measure 7-methylguanine in human DNA. DNA was digested to nucleotides and 7-methyl-2'-deoxyguanosine-3'-monophosphate (7-me-dGMP) was isolated from normal nucleotides using strong anion-exchange column chromatography. Overall the method gave 35-45% yield as measured with DNA methylated with tritiated dimethyl sulfate. Total white blood cell DNA from healthy non-smokers (n = 17) contained from 2.5 7-methylguanine residues/10(7) nucleotides, corrected for the losses in preparation. Among four patients sampled immediately after a total dose of 1050-2800 mg of dacarbazine or procarbazine, the mean adduct level was 57 7-methylguanine residues/10(7) nucleotides. As further method development, we also investigated the phosphorylation reaction by T4 polynucleotide kinase using dinucleotides containing 7-methylguanine and corresponding imidazole ring-opened products as substrates. We found that imidazole ring-opened dTpdG-Me is resistant to digestion with deoxyribonuclease I, snake venom phosphodiesterase and prostatic acid phosphatase. It is quantitatively phosphorylated at femtomolar levels. This method is shown to be suitable for the detection of 7-methylguanine in DNA, and is suggested to be the approach most suited to postlabelling large and labile 7-alkylguanines in DNA.

Abstract Several anticancer agents exert cytotoxicity by binding to DNA as their principle mechanism of action. However, limited information is available on the structural products of anticancer agents with DNA and measurements in humans have been carried out for cis-platin adducts only.

The 32P-postlabeling technique introduced by Randerath and co-workers has been particularly successful with stable and nonpolar DNA adducts, but the assay has not been used to any large extent to detect 7-alkylguanine derivatives. In the present communication, we have investigated the phosphorylation reaction by T4 polynucleotide kinase using 7-methyl-3'-dGMP, ring-opened 7-methyl-3'-dGMP and enzyme-digested methylated DNA as substrates. The methylated substrates were detected at femtomol (fmol) sensitivities. 7-methyl-3'-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less. It was shown that ring-opened 7-methyl-3'-dGMP was resistant to digestion with nuclease P1, making alkali-treatment and enzyme digestion of DNA possible approaches to the determination of 7-methylguanine in DNA.

The 32P-postlabelling technique introduced by Randerath and coworkers was used to investigate the efficiency of the phosphorylation reaction by T4 polynucleotide kinase using three synthesized adducts: 7-methyl-dGMP, ring-opened 7-methyl-dGMP and platinated dGpdG. The methylated substrates were detected at sub-fmol sensitivities. 7-Methyl-dGMP was quantitatively phosphorylated at these low concentrations. The efficiency of phosphorylation of the ring-opened product was less (about one order of magnitude) and that of Pt(dGpdG) about three orders of magnitude less. These results show that T4 polynucleotide kinase phosphorylation is an efficient reaction with 7-methyl-dGMP and with ring-opened 7-methyl-dGMP, even though in the latter case longer incubation times may have to be used to boost the reaction towards completion. By contrast, the low level of phosphorylation with Pt(dGpdG) does not appear encouraging for quantitative determination requiring a high sensitivity.

The antitumor activity of cisplatin (cis-diamminedichloroplatinum (II)) is thought to involve binding to cellular protein and nucleic acids (Roberts and Thomson 1979). The purpose of this study was to investigate using atomic absorption spectroscopy (AAS) the correlation between cisplatin dose and Pt concentrations in blood proteins (plasma proteins and hemoglobin) of cancer patients undergoing chemotherapy.

Two experiments were carried out by using atomic absorption spectroscopy on the stability and dose-dependence of cisplatin [cis-diamminedichloroplatinum (II)] binding to blood proteins and tissue DNA of male Han/Wistar rats. The dose-dependence was studied by injecting 17 rats i.p. either with cisplatin (4.4, 8.0 or 11.0 mg/kg) or 0.9% NaCl (controls). The Pt concentrations in blood proteins (plasma proteins and hemoglobin) and DNAs of different tissues (kidney, liver, lung and testis) were measured 24 h after the treatment. The binding of cisplatin to blood proteins and tissue DNAs correlated with each other and the dose. The stability was studied by treating 17 rats i.v. with 8.0 mg cisplatin/kg. The Pt concentrations in kidney, liver and lung DNA were determined 5 h, 1, 3 or 5 days after the treatment. The disappearance of Pt was faster in kidney DNA than in liver or lung DNA; in 5 days the Pt concentration in kidney DNA decreased by 63% while the Pt content in liver or lung decreased by 40%. Both of these experiments showed that the binding of cisplatin to kidney DNA exceeded the other tissue DNAs examined. Testicular DNA showed the lowest level of binding. The present animal data suggest that the platination level of blood proteins may be used as a measure of Pt concentration in tissue DNA.

The induction of hepatic peroxisome proliferation and drug metabolizing enzymes and of sister chromatid exchange (SCE) in lymphocytes was studied in male Han/Wistar rats after exposing them for 2 weeks to a commercial chlorophenolate formulation (Ky-5) (100mg/kg/ day), to 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD; 0.05–5 μg/kg/wk) and to the pure phenoxyacetic acids, 2,4-dichlorophenoxyacetic acid (2,4-D; 100 mg/kg/day) and 2-chloro-4-methylphenoxyacetic acid (MCPA; 100 mg/kg/day). The chlorophenolate formulation and pure 2,4-D and MCPA caused significant increases in the number of peroxisomes in liver cells, although the average size of peroxisomes was not affected, whereas the effect of even the highest dose of 2,3,7,8-TCDD remained small. This finding indicates that dioxin impurities do not account for the peroxisome proliferation induced by chlorophenolate. The relative weight of the liver increased significantly in rats treated with the chlorophenolate formulation and with 2,3,7,8-TCDD (5.0 and 0.5 μg/kg). The pattern of induction of xenobiotic metabolizing enzymes showed some differences between chlorophenolate treatment and 2,3,7,8-TCDD treatment. Furthermore, the effects of pure phenoxyacetic acids were different from that seen with chlorophenolate and 2,3,7,8-TCDD. The highest dose of 2,3,7,8-TCDD increased the frequency of SCE in circulating lymphocytes slightly, but significantly.

A study was conducted to determine the levels of cis-diamminedichloroplatinum (II) (cisplatin) in plasma proteins and hemoglobin of cancer patients after cisplatin chemotherapy. Thirty-seven cancer patients with different type of cancers (lung, esophageal, urinary tract, and testicular cancer, melanoma, osteosarcoma etc) received cisplatin 32–110 mg/m2 either as a single intravenous infusion or as infusions given on 5 consecutive days. Blood samples were classified according to time from previous cisplatin infusion. They included a total of 103 samples taken before the cisplatin infusion, immediately after infusion, 1, 2 or 3–5 days after infusion or 2–3, 4, or 5–7 weeks after infusion. Platinum (Pt) concentration in plasma proteins and hemoglobin was measured by atomic absorption spectroscopy (AAS). The data showed a correlation between the dose of cisplatin and the concentrations of Pt in plasma proteins and hemoglobin of cancer patients. Plasma proteins bound more cisplatin than hemoglobin, the respective maxima in the patients receiving > 50 mg/m2 being 27.7 and 1.6 ng/mg protein in samples drawn immediately after treatment. The kinetics of disappearance of Pt from plasma proteins showed several components; the initial half-life was about 5–7 days. The disappearance of Pt from hemoglobin showed a single component of a half-life of 12–14 days.

Experiments have been carried out to detemine the effect of different time intervals between the administration of X-irradiation (1200 rad) and cis-diamminedichloroplatinum (cis-DDP) (7 mg/kg) on the growth delay produced in three mouse tumors. The tumors used were the EMT6 tumor in BALB/c mice and the KHT and RIF-1 sarcomas in C3H mice. All tumors were grown intramuscularly in the gastrocnemius muscle and treatment was carried out at a mean tumor weight of 450 mg. Time to reach 2X (for KHT) or 4X (for EMT6 and RIF-1) treatment volume was used as the endpoint of response. The drug was administered by the intraperitoneal route either 24, 6, or 2 hr before radiation, immediately before the start of radiation, or 3, 6, or 24 hr after radiation. All irradiations were carried out in unanesthetized mice.The growth delays due to the drug alone were 2, 10, and 2 days for the EMT6, RIF-1, and KHT tumors, respectively. In the RIF-1 and KHT tumors, the combined modality groups tend to show longer growth delays than predicted by the addition of the growth delays for the single agents. For the EMT6 tumor, however, the trend is in the opposite direction. There is no particular timing between irradiation and drug administration which appears to produce consistently longer or shorter growth delays from system to system.

A new approach has been developed to determine the levels of cisplatin in different blood compartments of treated cancer patients. The cisplatin content of plasma, plasma proteins, red blood cells and white blood cell DNA can be measured by atomic absorption spectroscopy (AAS). The approximate levels of cisplatin were 10, 500 and 100 ng Pt/ml blood in plasma, plasma proteins and haemoglobin, respectively; in white blood cell DNA, the level of cisplatin was about 1 pg/microgram DNA. Preliminary data indicate that cancer patients have measurable amounts of cisplatin in their blood compartments. Furthermore, antibodies have been raised against cisplatin-DNA, with which 50% inhibition occurs at 50-100 fmol cisplatin. The detection limit is about 1-10 fmol cisplatin/microgram DNA. Enzyme immunoassay techniques will be used to detect cisplatin-DNA adducts in white blood cell DNA of cancer patients.