Effects of cytotoxic chemotherapeutic agents on split-dose repair in intestinal crypt cells.

PURPOSE: Many cancer chemotherapeutic agents interact with radiation to enhance the amount of radiation damage observed in both tumor and normal tissues. It is important to predict this interaction and to determine the effect of drug on sublethal damage repair. To evaluate for effects in rapid renewing normal tissues, the intestinal crypt cell in vivo assay is an excellent one to employ. These studies investigate the effect of eleven cancer chemotherapeutic drugs on split-dose repair in the intestinal crypt cell of the mouse. METHODS AND MATERIALS: LAF1 male mice, age 10-12 weeks, were exposed to whole-body irradiation with orthovoltage x-rays delivered as a single dose or as equally divided doses delivered with intervals between the two exposures of 2 to 24 h. In the experimental group, the cancer chemotherapeutic agent was administered intraperitoneally 2 h before the first radiation dose. At 3.6 days after the second irradiation, the mice were sacrificed; the jejunum was removed, fixed, and sectioned for light microscopy. The number of regenerating crypts were counted and corrected to represent the number of surviving cells per circumference. RESULTS: Of the eleven drugs tested, only carmustine eliminated split-dose repair. Cisplatin delayed repair, and methotrexate caused marked synchronization obliterating the observation of split-dose repair. CONCLUSIONS: Most cytotoxic chemotherapeutic agents do not inhibit sublethal damage repair in intestinal crypt cells when given 2 h before the first radiation exposure. Absence of the initial increase in survival seen with split-dose radiation is noted with carmustine and high-dose methotrexate.

Measurement of vascular permeability in spinal cord using Evans Blue spectrophotometry and correction for turbidity.

Vascular permeability can be visualized by Evans Blue (EB) extravasation and quantified by spectrophotometry after formamide extraction of the tissue. However, formamide extracts show significant turbidity, which may contribute to the total optical density at the wavelength of measurement (e.g., 620 lambda). We developed a simple method for estimating the component of the total optical density of a dyed specimen contributed by turbidity. Our method, which uses a determination of turbidity made at another point of the light spectrum (740 lambda), was more precise than two other EB quantification techniques. We therefore recommend it for individual correction of formamide extracts of spinal cord specimens. The application of this technique to the brain remains to be determined.

Radiation-induced decrease in nitric oxide synthase--containing nerves in the rat penis.

PURPOSE: Evaluate effect of prostatic irradiation on erectile function. MATERIALS AND METHODS: Forty-seven male adult rats were divided into three groups according to a single radiation dose to the prostate: control (no irradiation) (n = 15), 1,000 cGy (n = 15), and 2,000 cGy (n = 17). Five months after irradiation, rats underwent evaluation of penile vascularity and of erectile response to central and peripheral stimulation. After the study a proximal-shaft penile segment was obtained for staining. RESULTS: Histologic evaluation demonstrated that, with increasing radiation, the number of nitric oxide synthase-containing nerve fibers per penile segment decreased significantly: control, 225.6 +/- 9.7; 1,000 cGy, 156.3 +/- 12.0; 2,000 cGy, 85.8 +/- 10.1 (standard error of the mean). Maximal intracavernous pressure induced with electrostimulation decreased significantly with increasing radiation dose. After injection of papaverine, maximal intracavernous pressure was significantly decreased in only the 2,000-cGy group. CONCLUSION: A dose of 2,000 cGy over the prostatic bed induces erectile dysfunction by causing defects in the vascular supply of the erectile tissue and in the nerves and smooth muscle.

Lipid peroxidation does not appear to be a factor in late radiation injury of the cervical spinal cord of rats.

PURPOSE: We tested the role of lipid peroxidation in the demyelination and white matter necrosis associated with radiation injury of the central nervous system. METHODS AND MATERIALS: We irradiated the cervical spinal cords of female F344 rats (23 Gy) and assayed for the accumulation of the peroxidation byproducts malondialdehyde and hydroxyeicosatetraenoic acids, and for the consumption of the endogenous free radical scavengers vitamins E and C. We further tested the role of lipid peroxidation in radiation injury of the central nervous system by determining the sensitivity of the cervical spinal cord to radiation in rats on diets containing deficient, normal, and supplemental levels of the antioxidant vitamin E. Rats were placed on these diets at 4 weeks of age and irradiated (18.5-21.5 Gy) 16 weeks later. RESULTS: During the 5 months between irradiation and the onset of paralysis, no accumulation of peroxidation byproducts or consumption of endogenous scavengers was seen in the cervical spinal cords of the irradiated rats. The cervical spinal cords of some of the rats placed on the diets with deficient, normal, and supplemental levels of vitamin E were analyzed at the time of irradiation and contained 197 +/- 57, 501 +/- 19, and 717 +/- 35 pmol vitamin E/mg protein, respectively. Despite the statistical differences in these levels, the radiation sensitivity of the cervical spinal cord (ED50 for white matter necrosis) in rats receiving the three diets was not different (20.4, 20.7, and 20.6 Gy). CONCLUSION: These data do not support a role for free radical-induced lipid peroxidation in the white matter damage seen in radiation injury of the central nervous system.

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 cell's 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.

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.

In vivo radiobiology of heavy ions.

The radiobiology of heavy charged particles has been investigated with various animal systems in vivo at the Lawrence Berkeley Laboratory using the helium beam from the 184" synchrocyclotron and the carbon, neon, and argon beams from the BEVALAC. Tumor experiments were carried out using the R1 sarcoma in rats and the EMT6 mouse mammary carcinoma, comparing X rays, carbon ions, neon ions, and argon ions. In vivo normal tissue experiments have been carried out with a wide range of tissues including testis, bone marrow, intestinal crypt cells, lens of the eye, esophagus, lung, and the spinal cord. The induction of dominant lethal mutations after irradiation of the testis was assayed by in vitro embryo culture after in vivo irradiation. Experiments were also done with the Harderian gland tumor induction system.