William J. Bodell

Correlation of exposure time, concentration and incorporation of IdUrd in V-79 cells with radiation response☆

These experiments were designed to find the minimum concentration at which incorporation of and sensitization by IdUrd (Iododeoxyuridine) would occur and the effect of concentrations from .1 to 100 microM for exposures of 8 to 96 hr in cultured V-79 cells exposed to 137Cs gamma rays at 2 Gy per minute. At 0.1 microM thymidine replacement averaged 1% and the SER ranged from 1.1 to 1.28, significant at the 95% level. The maximum thymidine replacement was 49% after 48 hr exposure to 30 microM yielding an SER of 2.7. SER generally peaked after 72 hr of exposure. This cell line has an 8 hr cycle time in our hands and thus optimal sensitization would occur after 9 cell cycles. These ranges need testing in human cells in culture and in Phase I clinical trials.

Repair of O6-(2-chloroethyl)guanine mediates the biological effects of chloroethylnitrosoureas.

Chloroethylnitrosoureas (CENUs) are alkylating and crosslinking agents used for the treatment of human cancer; they are both mutagenic and carcinogenic. We compared the levels of induction of sister chromatid exchanges (SCEs) and the cytotoxicity of nitrosoureas that alkylate only with CENUs. CENUs are 200-fold more cytotoxic and induce SCEs with 45-fold greater efficiency than agents that do not crosslink; therefore, crosslinking is probably the most important molecular event that leads to cell death and induction of SCEs. The biological and biochemical properties of both human and rat brain tumor cells that are sensitive or resistant to the cytotoxic effects of CENUs have been investigated. CENUs induce SCEs in both sensitive and resistant cells, but to induce similar levels of SCEs, resistant cells must be treated with a 5- to 14-fold higher concentration of CENUs than are used to treat sensitive cells. Resistant cells have a higher cellular level of O6-methylguanine-DNA methyl transferase, increased repair of O6-methylguanine, and 50% fewer DNA interstrand crosslinks formed than do sensitive cells treated with the same concentration of CENU. Based on these findings, we propose that cellular resistance to the cytotoxic effects of CENUs is mediated by O6-methylguanine-DNA methyltransferase and that DNA repair may also modify the mutagenic and carcinogenic properties of CENUs.

Auger electron contribution to bromodeoxyuridine cellular radiosensitization

Halogenated thymidine analogs become incorporated into the DNA of proliferating cells during S-phase and may be used clinically to radiosensitize tumors that are otherwise poorly responsive to radiation. Although radiosensitization has been studied for years, mechanisms of radiosensitization are poorly understood. One possible mechanism involves the release of short range, high-LET, Auger electrons following photoelectric absorption of an X ray by the K-shell of the incorporated halogen. Such absorption occurs only with X ray energies slightly greater than the K-shell binding energy. We report the results of an experiment designed to measure this effect, in which cultured monolayers of Chinese hamster V79 cells, with 32% replacement of thymidine by bromodeoxyuridine (BUdR), were exposed to monoenergetic X rays just below (13.450 KeV) or above (13.490 KeV) the K-edge (13.475 KeV) of bromine. Enhancement ratios calculated in five different ways were slightly increased (3-12%) above the K-edge compared to below. However, only a calculation using a linear-quadratic fit to the data and a surviving fraction of 0.01 demonstrated a statistically significant increased enhancement ratio (12%) above the K-edge. We conclude that Auger electrons produced following photoelectric absorption of X rays by the K-shell of bromine contribute minimally to observed BUdR cellular radiosensitization.

Molecular dosimetry for sister-chromatid exchange induction and cytotoxicity by monofunctional and bifunctional alkylating agents

The induction of sister-chromatid exchanges (SCEs) and cytotoxicity in 9L cells treated with monofunctional and bifunctional alkylating agents has been investigated. Three classes of monofunctional and bifunctional agents were studied: nitrosoureas, mustards and epoxides. Independent of class the bifunctional agents were 55-630-fold more effective at inducing SCEs and 300-2400-fold more effective at inducing cellular cytotoxicity than the corresponding monofunctional agents. Comparing the induction of SCEs and cytotoxicity by these agents showed that these two cellular responses to DNA damage are highly correlated. The extent of DNA alkylation in cells treated with 1-ethyl-1-nitrosourea (ENU) or 1-(2-chloro-ethyl)-1-nitrosourea (CNU) was similar indicating that the increased effectiveness of CNU to induce SCEs and cytotoxicity was not due to increased DNA alkylation. Molecular dosimetry calculations indicate that for CNU and ENU treatment of 9L cells there are 116 and 8500 alkylations per SCE induced and 2.6 x 10(4) and 4.6 x 10(6) alkylations at the dose required to reduce survival of 9L cells by 90%. Comparison of the DNA alkylation products produced by CNU and ENU treatment of 9L cells suggests that the formation of the intrastrand crosslink N7-bis(guanyl)ethane and the interstrand crosslink 1-(3-deoxycytidyl)-2-(1-deoxyguanosinyl)ethane by CNU is responsible for the increased effectiveness of CNU treatment at both induction of SCEs and cytotoxicity.

Relationships among DNA adducts, micronuclei, and fitness parameters in Xenopus laevis exposed to benzo[a]pyrene

Abstract We investigated whether hepatic DNA adducts, erythrocytic micronuclei, wet weight, developmental stage, wet weight at metamorphosis, and time to metamorphosis changed in larval Xenopus laevis exposed to varied doses of benzo[a]pyrene (B[a]P). Using 32P-postlabeling, we observed relative DNA adduct levels of 0 to 13.7 × 10−7 following continuous exposure to 0 to 496 nM B[a]P for 12 days and relative levels of 0 to 10 × 10−7 after exposure to 248 nM B[a]P over a range of 0 to 16 days. Mean numbers of micronuclei were 1.7, 6.3, and 16.4 1000 red blood cells after exposure to 0, 31, and 248 nM B[a]P, respectively, for 14 days. Micronuclei also ranged from 1.3 to 120.5 1000 red blood cells following exposure to 248 nM B[a]P over a range of 0 to 16 days. Comparatively, levels of both DNA adducts and micronuclei were greatly reduced in animals exposed previously to 31 and 248 nM B[a]P, but assayed at metamorphosis. Larvae exposed to 248 nM B[a]P for 14 days took approximately 4 days longer to metamorphose than unexposed larvae. This increased time to metamorphosis was associated with increased DNA adducts and micronuclei in larvae exposed to 248 nM B[a]P. However, DNA adducts and micronuclei also increased in larvae exposed to 31 nM B[a]P, while time to metamorphosis did not. Larval wet weight was reduced by as much as 44% immediately following exposure to B[a]P. However, there was no effect of exposure on wet weight at metamorphosis. Exposed animals were up to 2 developmental stages younger than unexposed animals in one experiment, but differences among exposed and unexposed animals were less distinct in a second experiment. These studies suggest that DNA adducts and micronuclei can be sensitive measures of sublethal DNA damage, as well as possible short-term indicators of indirect effects on fitness in amphibians.

Iododeoxyuridine incorporation and radiosensitization in three human tumor cell lines

Iododeoxyuridine is a halogenated pyrimidine and non-hypoxic cell radiosensitizer currently being used in clinical trials. The amount of radiosensitization by IdUrd is related to the amount of incorporation of the drug into a cells DNA. These experiments were carried out in three human tumor cell lines (lung, glioma, and melanoma) in monolayer culture exposed to concentrations of IdUrd from 0.1-10 microM for one and three cell cycles before irradiation to determine incorporation and sensitization as a function of drug exposure. Except for the lung cell line, which required greater than 1 microM IdUrd, these cells demonstrate radiosensitization when exposed to 0.1 microM or greater of IdUrd. Maximum sensitization occurred at 10 microM IdUrd for all the cell lines at three cell cycles. The percent thymidine replacement by IdUrd increased with increasing concentrations, but was cell line dependent. Maximum percent replacement occurred at 10 microM at three cell cycles for all the cell lines: lung = 22.4%, glioma = 32.0%, and melanoma = 39.1%. The relationships between percent thymidine replacement and sensitization are not identical across these human tumor cell lines. If IdUrd is going to be a successful radiosensitizer in clinical trials, sustained plasma levels of 10 microM or greater for at least three cell cycles should be achieved during irradiation. This may be best accomplished with repeated short exposures to IdUrd (three cell cycles or approximately 4 days in these cell lines) every 1-2 weeks during radiation. Measurements of thymidine replacement in a tumor biopsy should be attempted prior to radiation to develop a predictive assay for radiosensitization.

Repair of DNA alkylation products formed in 9L cell lines treated with 1-(2-chloroethyl)-1-nitrosourea.

The purpose of this study has been to measure the formation and repair of individual DNA alkylation products in 9L, 9L-2 and BTRC-19 cell lines after treatment with 1-(2-chloroethyl)-1-nitrosourea (CNU). The levels of seven DNA adducts N7-(2-hydroxyethyl)-guanine, N7-(2-chloroethyl)-guanine; 1,2-(diguan-7-yl)-ethane, N1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N1-2-deoxyguanosinyl), 2-(N3-2-deoxycytidyl)-ethane, O(6)-(2-hydroxyethyl)-2-deoxyguanosine and phosphotriesters were separated by HPLC and quantified by liquid scintillation counting. The levels of N7-(2-hydroxyethyl)-guanine, N7-(2-chloroethyl)-guanine; O(6)-(2-hydroxyethyl)-2-deoxyguanosine and phosphotriesters were not significantly different in the three glioma lines. Furthermore, comparison of the levels of these products in treated cells with the levels formed in purified DNA suggest that they were not actively repaired over the 6h interval. The levels of 1,2-(diguan-7-yl)-ethane and N1-(2-hydroxyethyl)-2-deoxyguanosine were reduced in 9L-2 and significantly reduced in BTRC-19 (P = 0.003) compared to 9L. Analysis of the data suggests that the reduction in the level of N1-(2-hydroxyethyl)-2-deoxyguanosine was due to repair of its precursor O(6)-ClEtdG by O(6)-alkylguanine-DNA-alkyltransferase (AGT). The level of the crosslinked product 1-(N1-2-deoxyguanosinyl), 2-(N3-2-deoxycytidyl)-ethane was significantly reduced (P < 0.001) in both 9L-2 and BTRC-19 as compared to 9L. Reduction in the level of 1-(N1-2-deoxyguanosinyl), 2-(N3-2-deoxycytidyl)-ethane in 9L-2 and BTRC-19 are consistent with repair of the precursor alkylation product O(6)-ClEtdG by AGT. This study demonstrates that there are very significant differences in the rates of removal of individual DNA adducts formed by CNU treatment of the glioma cell lines.

Comparison of sister-chromatid exchange induction caused by nitrosoureas that alkylate or alkylate and crosslink DNA.

We have investigated the induction of sister-chromatid exchanges (SCEs) in 9L rat brain tumor cells treated with the alkylating agent 1-ethyl-1-nitrosourea (ENU) and 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea (ACNU), an agent that both alkylates and crosslinks DNA. Induction of SCEs by ACNU was found to be 143-fold greater than for ENU. However, on an equimolar basis, the alkylation of DNA by 14C-ACNU was approximately 3.2-fold higher than for 14C-ENU. After correction for this difference was made, the induction of SCEs by ACNU was calculated to be 45-fold greater than for ENU. While DNA alkylation products formed by ACNU and ENU are similar, the chloroethyl alkylation product(s) of ACNU can form DNA-interstrand crosslinks; the ethyl alkylation product(s) of ENU cannot. Based on these findings, we propose that the increased induction of SCEs caused by ACNU is a result of the formation of DNA interstrand crosslinks.

Levels of N7-(2-hydroxyethyl)guanine as a molecular dosimeter of drug delivery to human brain tumors.

The level of N7-(2-hydroxyethyl)guanine (N7-HOEtG), one of the DNA alkylation products formed by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment, was measured in human brain tumor samples by high performance liquid chromatography with electrochemical detection. The tumors from 6 recurrent chemotherapy-naive patients with recurrent glioblastoma multiforme were analyzed as controls. The mean level of N7-HOEtG in DNA of these specimens was 0.42 pmol/mg DNA. Samples were also obtained from a patient with a recurrent glioblastoma multiforme after direct intratumoral therapy with BCNU in ethanol (DTI-015). The levels of N7-HOEtG in the samples distal, medial, and adjacent to the site of injection were 0.8, 2.6, and 369.5 pmol/mg DNA, respectively. Comparison of the level of N7-HOEtG detected in the distal sample after injection with BCNU in ethanol with the mean level of the untreated samples indicated that it was not sufficiently different to be ruled out as a chance occurrence. Comparison of the levels of N7-HOEtG in the medial and adjacent brain tumor samples with the mean level of the control samples showed values that were approximately 6- and 879-fold higher. These results demonstrate that intratumoral administration of BCNU in ethanol produces significant levels of DNA alkylation and suggest that DNA adduct measurements provide a unique molecular dosimeter to evaluate delivery of alkylating agents to brain tumors.

Levels and distribution of BCNU in GBM tumors following intratumoral injection of DTI-015 (BCNU-ethanol).

The alkylation products formed by in vitro treatment of DNA with tritium-labeled 1,3-bis(2-chloroethyl)-1-nitrosourea ((3)H-BCNU) were identified and quantified. Twelve adducts were resolved by high-performance liquid chromatography (HPLC). The principal DNA adducts formed by BCNU treatment corresponded to N-7-(2-hydroxyethyl)guanine (N7-HOEtG) (26%), N-7-(2-chloroethyl)guanine (15%), and phosphotriesters (19%). In addition, several minor products were identified as 1,2-(diguan-7-yl)ethane, N-1-(2-hydroxyethyl)-2-deoxyguanosine, 1-(N-1-2-deoxyguanosinyl), 2-(N-3-2-deoxycytidyl)ethane cross-link, and O-6-(2-hydroxyethyl)-2-deoxyguanosine, and individually they represented 1% to 5% of the total alkylation. An HPLC-electrochemical method was applied to quantify the levels of N7-HOEtG in samples treated with BCNU. Treatment of either purified DNA or U87MG cells with various amounts of BCNU produced a linear increase in the amount of N7-HOEtG. These results demonstrated that the levels of N7-HOEtG formed by BCNU treatment could be used as a molecular dosimeter of BCNU treatment dose. We measured the levels of N7-HOEtG in DNA isolated from tumor samples taken from four patients with GBM tumors following stereotactic intratumoral injection with DTI-015 (BCNU-ethanol). The level of N7-HOEtG in these samples ranged from 14.7 to 121.9 micromol N7-HOETG/mol DNA within 1 cm of the site of injection. As the distance from the site of injection increased, the levels of N7-HOEtG in tumor DNA decreased. In two of the samples, the levels of N7-HOEtG were 0.2 to 0.3 micromol N7-HOETG/mol DNA at 3.5 to 3.9 cm from the site of injection, demonstrating significant distribution of BCNU in the tumor. The levels of N7-HOEtG in these tumor samples corresponded to BCNU treatment concentrations of 0.02 to 43.0 mM. These studies demonstrate that stereotactic intratumoral injection of DTI-015 into human GBM tumors produces high concentrations of BCNU up to 2.5 cm from the site of injection in some of the tumors. These observations suggest that intratumoral injection of DTI-015 may be of benefit in the treatment of primary and recurrent GBM tumors.