Alan D. Conger

Is age-dependent genetic damage in seeds caused by free radicals?*

Abstract In dry seeds of several kinds stored for up to 48 yr, no significant changes in electron spin resonance patterns (neither signal amplitudes nor shapes) vs. age were found, although four classes of signals were observed. This result is counter to some crude notions of how genetic damage from aging of seeds might arise from free radical reactions, and differs from ESR observations related to radiation-induced damage.

A correlation of seedling height and chromosomal damage in irradiated barley seeds

Abstract The most commonly used measurement of radiation damage to seeds is seedling height, the mean height a lot of seeds attains at some time during exponential growth. If planted immediately post-irradiation, seeds of a dose lot give a normal height distribution, but if stored before planting give very abnormal and even bimodal height distributions. By within-seed comparisons of chromosome abnormality (from roots excised at 24–36 hr) with height (attained by 7–9 days) in irradiated barley seeds, it is shown that damage to height and to chromosomes are closely correlated, even within a treatment in which great heterogeneity occurs. The two effects have equal radiosensitivity, but different shoulders to their dose curves. Seedling height is not depressed until 25–30 per cent of the cells bear chromosomal abnormalities. The heterogeneity observed is not due to a between-seed heterogeneity in dose or in oxygen content, and probably not in moisture. These experiments show that the heterogeneity arises from factors that operate on post-irradiation (indirect) storage damage, but are without effect on during-irradiation damage (direct).

Real chromatid deletions versus gaps.

Abstract The validity of previous scoring of radiation-induced chromatid deletions detected at metaphase has been brought into serious question by Revell in recent years. He finds in Vicia that only 1 20 to 1 100 of the apparent chromatid deletions are real, the remaining large fraction are achromatic lesions or “gaps”. Here, this problem was studied in Tradescantia microspores, γ-irradiated in late interphase (S or perhaps G 2 ), by analysis of the chromatid types of aberrations at metaphase and at anaphase. A real chromatid deletion will release its fragment at anaphase, the gap will not. The scoring validity of metaphase-detected chromatid deletions was established by observation, at the same dose, of an equal frequency of free fragments at anaphase. Metaphase scoring of all other aberration types was similarly confirmed, and yield per rad agreed with previous metaphase scoring. The frequency of gaps was at the most but a third that of the confirmed real chromatid deletions, actually probably less. (Radiation was delivered at low intensity (50 rad/h), which, on Revells hypothesis, should have maximized the ratio of gap to chromatid deletion production.) Reasons for the 60- to 300-fold disagreement ( 1 3 versus 20 1 to 100 1 ) in scoring of gaps in Vicia and in Tradescantia are considered. Differences in microscopic detection, staining methods, and stages irradiated are unlikelily as major contributors to the discrepancy; intrinsic species or possibly cell type differences are left as the most likely, though unexplained, reasons.

WR-2721 Entry into the Brain across a Modified Blood-Brain Barrier

Radioprotection of the CNS by WR-2721 has not been possible because of its inability to cross the blood-brain barrier (BBB) and so gain access to the neural tissue. Modification of the BBB using hypertonic arabinose (1.8 m), injected via the internal carotid artery (ica), permitted entry of ip-injected [14C]WR-2721 into the ipsilateral cerebral hemisphere. The BBB-modified hemisphere had a 5.34-fold increased uptake compared to nonmodified controls. Delivery as a bolus via the ica further enhanced uptake after BBB opening; WR-2721 was 3.73 times greater than by ip injection. A 20-fold increase of WR-2721 brain uptake has been calculated for ica administration with the BBB opened as compared to the ip route without BBB modification. Toxicity of ip-administered WR-2721 with the BBB open was only 1.4 times greater than non-modified controls and 1.96 times more toxic when delivered via the ica. These data demonstrate significant uptake of WR-2721 into the CNS, a previously unprotected organ, and provide a model for future radioprotective studies.

Effect of chromosomal breaks induced by X-irradiation on the number of mesosomes and the cytoplasmic organization of Streptococcus faecalis

Abstract A model which explains mesosome formation via a contraction of the cytoplasm and nucleoid when bacteria are physiologically disturbed was tested by: (1) X-irradiation of unfixed cells of Streptococcus faecalis to produce chromosomal breaks and to remove DNA attached to the cell membrane; (2) subsequent determination of the number of irradiated cells in which mesosomes (using electron microscopy) and central density changes (using phase-contrast microscopy) could be visualized after fixative was added. Results obtained by exposure of cells to doses up to 1100 krads before fixation indicated that: (1) the number of cells with central mesosomes was reduced proportional to the decrease in the molecular weight of the DNA due to double-strand breaks: (2) the number of cells with total (central plus peripheral) mesosomes and the number of cells with peripheral mesosomes were both reduced proportional to the removal of DNA attached to the cell membrane (M band); (3) the nucleoid became more diffusely organized. Exposure of cells to doses greater than 1100 krads before fixation resulted in: (1) an increase in the number of cells with central and peripheral mesosomes (compared to cells exposed to lower dosages); (2) a return to the centralized, dense nucleoid seen in unirradiated cells. These results suggest that mesosomes are formed when localized sites on the cell membrane are pulled from close contact with the cell wall into the cytoplasm by the action of a cross-linking fixative via the aggregation of intracytoplasmic components such as DNA. This model considers the attachment of DNA and/or other cytoplasmic components to the membrane as an intrinsic part of its mechanism. The formation of central and peripheral mesosomes in unirradiated and X-irradiated cells are contrasted.

Effect of WR-2721 on fetal development in the rat

The radioprotector WR-2721 has been shown to radioprotect all tissues studied except the central nervous system. However, it has not yet been used to radioprotect the fetus. In this study we determined that [14C]WR-2721 injected into pregnant rats quickly passed the placenta and was concentrated by the fetus. In addition, we evaluated the toxicity of WR-2721 (2.5-600 mg/kg) to pregnant rats and their fetuses during the period of major organogenesis at Days 9, 11, and 14 postconception. Pregnant animals were only slightly more (10%) sensitive (LD50, 580 mg/kg) to WR-2721 than nonpregnant cohort animals (LD50, 640 mg/kg). At concentrations of 50 mg/kg or less there was a small but statistically significant increase in fetal deaths, while at doses greater than 300 mg/kg a larger degree of fetal mortality occurred. Maximal fetal weight loss, to about 84% of control, was found at 500 mg/kg. No changes in head dimensions or gross malformations of the surviving fetuses were detected at any time or concentration. Of all the parameters measured in this study none demonstrated a predilection for any specific period of major organogenesis. The results of this study indicate that while WR-2721 demonstrates a dose-related embryotoxicity it is not teratogenic.

A test for a magnetic effect in irradiated seeds

Abstract A magnetic effect on radiation damage in seeds was sought. Dry dormant barley seeds were X-irradiated, and exposed to uniform permanent magnetic fields (4 to 10 kG) or to dummy magnets (no magnet) in dry air at room temperature. Seeds were exposed in three experiments to magnetic fields for ( a ) 1 day post irradiation, ( b ) during irradiation and for 4 days post irradiation and ( c ) during and for 4 days post irradiation and during the 2-hr period of water soaking to start germination. These are periods during which many other modifiers profoundly affect radiation damage, and when radiation-induced free radicals are present in the seeds. Seeds were grown for about 10 to 12 days, and height of the first leaf (the well-known seedling height) used as criterion of radiation damage. Seedling height was not affected by the magnetic treatments.

Reduction of Radiation-Induced Hair Loss by Topical Application of Radioprotectors

AbstractOur transdermal permeation studies of radioprotectors in permeation-enhancing vehicles led us to hypothesize that radiation-induced hair loss could be reduced by topical application of radioprotectors. In the present study we used a hair regrowth assay in “plucked” mice to measure the radioprotective effectiveness of WR-1065, cysteine and TEMPOL when they were dissolved in a variety of vehicles, i.e., saline, dimethyl formamide (DMF), propylene glycol (PG), dimethyl sulfoxide (DMSO), ethanol. Protector effectiveness varied with radiation dose and vehicles. At 600 R, WR-1065 or cysteine, in any vehicle, reduced radiation-induced baldness scores; at 800 R, only WR-1065 in DMF and cysteine in saline, produced some protection. TEMPOL in ethanol produced protection at both the 600 and 800 R. Results suggest that suitably chosen topical application of protector/vehicle combinations can reduce radiation-induced hair loss.

Transpiacental uptake of WR 2721 by the rat embryo

On day 14 post-conception, near the end of the period of major organogenesis, pregnant rats were injected intravenously or intraperitoneally with WR 2721 spiked with 14C-WR 2721. The radioprotectant was shown to cross the placenta rapidly when administered by either route, and the concentration of WR 2721 in the embryos, placentae, and maternal blood plasma was determined during the period 5 to 90 minutes following administration. The concentration of WR 2721 increased continuously in the embryos during this period and did so against a decreasing concentration in the maternal blood. Injection of WR 2721 at 100 mg/kg of maternal body weight resulted in the presence of 8-9-mg/kg embryo weight; this embryo level is about 1/2 the injected dose of WR 2721 currently being used in human radiotherapy trials, that is, 20 mg/kg (740 mg/m2) body weight. Previous toxicity studies of 9, 11, and 14 day rat embryos have shown that this 100 mg/kg dose is much below the level which produces embryotoxic effects.