Ethylphosphorothioic Acid Research Articles

Indian Indigenous Fruits as Radioprotective Agents: Past, Present and Future

Dietary agents with beneficial effects like free radical scavenging, antioxidant and immunomodulatory effects are being recognized as useful and have been investigated for their radioprotective properties. Studies in these lines have shown that the fruits of Aegle marmelos (stone apple or bael), Emblica officinalis or Phyllanthus emblica (Indian gooseberry/amla), Eugenia jambolana or Syzygium jambolana (black plum/jamun), Mangifera indica (mango) and Grewia asiatica (phalsa or falsa) that are originally reported to be indigenous to India have been investigated for their usefulness as radioprotective agents. The objective of this review is to summarize the beneficial effects of the Indian indigenous fruits, stone apple, mango, Indian gooseberry, black plum, and phalsa, in mitigating radiation-induced side effects, emphasize the underlying mechanism of action for the beneficial effects and address aspects that merit detail investigations for these fruits to move towards clinical application in the near future. The authors data-mined Google Scholar, PubMed, Embase, and the Cochrane Library for publications in the field from 1981 up to July 2020. The focus was on the radioprotection and the mechanism responsible for the beneficial effects, and accordingly, the articles were collated and analyzed. This article emphasizes the usefulness of stone apple, mango, Indian gooseberry, black plum, and phalsa as radioprotective agents. From a mechanistic view, reports are suggestive that the beneficial effects are mediated by triggering free radical scavenging, antioxidant, anti-mutagenic and anti-inflammatory effects. For the first time, this review addresses the beneficial effects of mango, Indian gooseberry, black plum, stone apple and phalsa as radioprotective agents. The authors suggest that future studies should be directed at understanding the selective radioprotective effects with tumor-bearing laboratory animals to understand their usefulness as radioprotective drug/s during radiotherapy and as a food supplement to protect people from getting exposed to low doses of radiation in occupational settings. Phase I clinical trial studies are also required to ascertain the optimal dose and the schedule to be followed with the standardized extract of these fruits. The most important aspect is that these fruits, being a part of the diet, have been consumed since the beginning of mankind, are non-toxic, possess diverse medicinal properties, have easy acceptability, all of which will help take research forward and be of benefit to patients, occupational workers, agro-based sectors and pharma industries.

Hydrogen-rich saline attenuates radiation-induced male germ cell loss in mice through reducing hydroxyl radicals

Our recent studies suggest that H2 (hydrogen) has a potential as a novel radioprotector without known toxic side effects. The present study was designed to examine the underlying radioprotective mechanism of H2 and its protective role on irradiated germ cells. Produced by the Fenton reaction and radiolysis of H2O, hydroxyl radicals (•OH) were identified as the free radical species that were reduced by H2. We used a H2 microelectrode to dynamically detect H2 concentration in vivo, and found H2 significantly reduced in situ fluorescence intensity of hydroxyphenyl fluorescein; however, as we treated the mice with H2 after irradiation, the decrease is not significant. We found that pre-treatment of H2 to IR (ionizing radiation) significantly suppressed the reaction of •OH and the cellular macromolecules which caused lipid peroxidation, protein carbonyl and oxidatively damaged DNA. The radioprotective effect of H2 on male germ cells was supported by ameliorated apoptotic findings examined by morphological changes and TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling) in testicular tissue, and by preserved viability of stem spermatogonia examined for testicular histological parameters, daily sperm production and sperm quality; we used WR-2721 [S-2-(3-aminopropylamino)ethyl phosphorothioic acid] as a reference compound. Our results represent the first in vivo evidence in support of a radioprotective role of H2 by neutralizing •OH in irradiated tissue with no side effects.

Abstract 2498: Resveratrol administration increases SOD2 and reduces oxidative stress to DNA

Abstract Mitochondrial manganese superoxide dismutase (SOD2) activation is a downstream product after stimulation via a variety of signaling pathways from a diversity stimulating agents. Thiols including amifostine (S-2-[3-aminopropylamino]ethylphosphorothioic acid) mesna (sodium-2-mercaptoethanesulfonate), NAC (N-acetyl-L-cysteine) and captopril ([S]-1-[3-mercapto-2-methyl-1-oxo-propyl]-L-proline), are effective inducers of SOD2. Non-thiols including estrogen, cerium oxide, low doses of x-rays, and resveratrol (3,4’,5-trihydroxystilbene) have been shown to induce SOD2 under specific treatment conditions. Studies with resveratrol typically involve continuous exposure of cells for several days before determining the effectiveness of the agent as a radioprotector. In studies reported here, SA-NH murine sarcoma tumor cells established for growth in culture were exposed to a single dose of 50μM resveratrol and analyzed for SOD2 activity from 2hr to 28hr post exposure. Early indications suggest that cells induce SOD2 earlier than typically seen for thiols and other oxidative stress modifiers. Resveratrol increases levels of SOD2 by a factor of 3 compared to controls which is similar to levels observed with thiol exposure. While thiols induce SOD2 via the NFκB pathway, resveratrol acts through the FOXO3a pathway. Studies continue to examine SOD2 activity after exposure to acute exposures resveratrol, amifostine, and x-rays. This work was supported by DOE grant DE-SC0001271 (D.J.G.) and NIH grant RO1-CA132998 (D.J.G.). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2498. doi:10.1158/1538-7445.AM2011-2498

Immobilization of aminothiols on poly(oxyethylene H‐phosphonate)s and poly(oxyethylene phosphate)s—An approach to polymeric protective agents for radiotherapy of cancer

AbstractThis study describes the synthesis and characterization of polymer complexes constructed from the radioprotective agent S‐2(3‐aminopropylamino) ethylphosphorothioic acid dihydrate (amifostine or WR‐2721), applied in the radiation cancer treatment, and biodegradable poly(oxyethylene H‐phosphonate), poly(hydroxyoxyethylene phosphate), or poly(methyloxyethylene phosphate). The immobilization of another radioprotector, used in cancer radiotherapy, 1‐(3‐aminopropyl)aminoethanethiol (WR‐1065) on the same polymers is also achieved through a covalent bond (Atherton‐Todd reaction coupling), ionic bond, and physical complexation, respectively. The structure of the complexes formed is elucidated by 1H‐ 13C‐, 31P NMR and FTIR spectroscopy. © 2007 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 45: 1349–1363, 2007

Amifostine Protects Against Cisplatin-Induced Nephrotoxicity in a Child with Medulloblastoma

Amifostine (WR-2721, Ethyol) (S-2-(3-aminopropylamino)-ethylphosphorothioic acid) protects normal tissue from the cytotoxic damage induced by radiation and by chemotherapy. Several studies in adults have shown that amifostine reduces the cumulative renal toxicities associated with cisplatin, with no reduction in its anti-tumor efficacy [1, 2]. In the pediatric age group, amifostine has also been shown to be well tolerated and to ameliorate the myelotoxicity from alkylating agents and from cisplatin, in a variety of solid tumors [3, 4]. We describe the first reported case of the use of amifostine to protect against cisplatin-induced nephrotoxicity in a child with medulloblastoma who had a solitary kidney. A 7-year-old boy was referred with complaints of headache, vomiting, and diplopia. Magnetic resonance imaging (MRI) scan of the brain showed an enhancing tumor 3 cm in diameter located in the cerebellar vermis, invading the right cerebellar hemisphere, and partially filling the fourth ventricle, with moderate internal hydrocephalus. MRI scan of the spine demonstrated gross nodular seedlings in the thoraco-lumbar subarachnoid space. The tumor was staged as T2M3 according to the classification by Chang et al. [5]. The patient underwent posterior fossa decompression and a partial resection of the tumor mass with less than 50% residual disease demonstrated postoperatively. Histopathological analysis confirmed the diagnosis of medulloblastoma. Within 28 days of surgery, he began radiation therapy and received 3780 cGy to the craniospinal axis with a 1980-cGy boost to the local tumor site (a total radiation dose to the tumor site of 5760 cGy). Daily fractions of

Differential antigenotoxic and cytoprotective effect of amifostine in idarubicin‐treated mice

In this study we evaluated the antigenotoxic and cytoprotective capabilities of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid (amifostine)] in different normal tissues of BALB/c mice treated with idarubicin [4-demethoxydaunorubicin (IDA)]. The aminothiol WR-2721 is a pro-drug that requires dephosphorylation to its active metabolite WR-1065, to produce selectively cytoprotective activity in normal tissues exposed to radio- and chemotherapeutic agents, without protecting malignant tissues. IDA is an effective chemotherapeutic agent against hematological diseases, but produces a broad spectrum of toxicity in nontumoral cells. Animals were injected intravenously with WR-2721 (250 mg/kg) or IDA (6 mg/kg) and WR-2721/IDA. Micronuclei frequency in bone marrow was measured 24 and 48 hr after initiation of the treatments. The IDA-treated group showed increased levels of micronuclei. However, the WR-2721- and WR-2721/IDA-treated groups did not show differences from the controls. Genetic damage was evaluated by alkaline single-cell gel electrophoresis at 24-hr posttreatments. Important DNA damage was observed in liver, spleen, and peripheral blood cells of mice treated with IDA. The presence of WR-2721 diminished that damaging effect only in liver cells. The apoptotic index was measured in liver and spleen tissues by the TUNEL assay 14 and 24 hr after treatment. In liver we observed an increased percentage of apoptotic cells at 24 hr for the IDA-treated group, whereas the WR-2721 and WR-2721/IDA groups remained at low levels. Splenic cells treated with IDA and WR-2721/IDA showed increased DNA fragmentation levels at any time. In conclusion, WR-2721 has a tissue-specific antigenotoxic and cytoprotective effect in IDA-treated mice using these experimental conditions.

Substitution reactions of [Pt(dien)Cl]+, [Pt(dien)(GSMe)]2+, cis-[PtCl2(NH3)2] and cis-[Pt(NH3)2(GSMe)2]2+ (GSMe =S-methylglutathione) with some sulfur-bonding chemoprotective agents

Sulfur-containing compounds are used as rescue agents to protect normal tissue from the toxic side effects of cis-[PtCl2(NH3)2] (cisplatin) without affecting its anti-tumour activity. Reactions of the model complexes [Pt(dien)Cl]+ (1) and [Pt(dien)(GSMe)]2+ (2) (GSMe = S-methylglutathione) with the rescue agents glutathione (GSH), thiourea, thiosulfate, and diethyldithiocarbamate (DDTC) have been studied in a 1.0 mol dm−3 aqueous perchlorate medium at 37 °C and pH 7.30 using stopped-flow and conventional UV/VIS spectrophotometry. The reactions of 1 with 2-mercaptoethanesulfonate (mesna) and 2-mercaptoethylamine (cysteamine), which is the parent compound of 2-(3-aminopropylamino)ethylphosphorothioic acid (WR2721), and of cis-[Pt(NH3)2(GSMe)2]2+ with thiosulfate have also been studied. The reactions between cisplatin and DDTC, thiourea and thiosulfate were investigated in an unbuffered 0.10–0.05 mol dm−3 chloride medium at 37 °C. All reactions follow the rate law: −d[complex]/dt = k2[Pt(II)][Nu] where k2 denotes a second-order rate constant and [Nu] is the total concentration of nucleophile. The rate law indicates that the reactions proceed via a direct nucleophilic substitution pathway. The rescue agents display a relatively narrow range of reactivity towards 1, mesna < Hcysteamine < GSH < thiourea < thiosulfate < DDTC with rate constants k2 of 0.156 ± 0.001, 0.22 ± 0.01, 0.237 ± 0.003, 1.17 ± 0.01, 5.57 ± 0.04, and 8.0 ± 0.1 mol−1 dm3 s−1, respectively. The sequence is consistent with the report that DDTC is the most effective rescue agent discovered so far when administered after cisplatin. The activation parameters ΔH≠ and ΔS≠ have been determined and the products of the reaction of 1 with the rescue agents have been characterised.

Novel Approach to In Vivo Screening for Radioprotective Activity in Whole Mice: In Vivo Electron Spin Resonance Study Probing the Redox Reaction of Nitroxyl

Previously, we reported that X-irradiation enhanced the signal decay of a spin probe injected into whole mice measured by in vivo ESR, and that the observed enhancement was suppressed by the pre-administration of cysteamine, a radioprotector [Miura, Y., Anzai, K., Urano, S. and Ozawa, T. (1997) Free Rad. Biol. Med. 23: 533-540]. In the present study, the suppression activity of the X-ray-induced increase in the ESR signal decay rate (termed suppression index, SI) was measured for several radioprotectors: 5-hydroxytryptamine (5-HT), S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721), 4-hydroxy-2,2,6,6-tetramethyl-piperidine-N-oxyl (TEMPOL), cimetidine, interleukin-1 beta (IL-1 beta) and stem cell factor (SCF). The enhancement of the ESR signal decay of carbamoyl-PROXYL due to X-irradiation was suppressed by a treatment with all of the radioprotectors examined, showing positive SI values. However, a dose-dependency of 5-HT or WR-2721 was not observed, suggesting that several mechanisms exist for radioprotection and a modification of the signal decay rate. Although the in vivo ESR system cannot be used in place of the 30-day survival method for the assessment of radioprotectors, this system might be applicable to in vivo, non-invasive screening prior to using the 30-day survival method.

Prevention of radiation-induced mammary tumours in rats by combined use of WR-2721 and tamoxifen

Purpose : This investigation evaluated the inhibitory effect of S-2- (3-aminopropylamino)-ethylphosphorothioic acid (WR-2721) against the initiation of mammary tumourigenesis by irradiation, and the antipromotion activity of tamoxifen in the development of radiation-initiated mammary tumours. Materials and methods : Lactating rats were injected with WR-2721 and then irradiated with γ-rays (1.5 Gy) at day 21 of lactation. The rats were divided into three groups 1 month after irradiation and were implanted with a pellet either of cholesterol as an inert control, diethylstilbestrol (DES) as a tumour-promoting agent, or DES combined with tamoxifen. For the control experiments, non-irradiated and irradiated rats receiving saline instead of WR-2721 were treated with a pellet by the same procedures. Results : The highest incidence (85%) for tumourigenesis of mammary glands was observed in the irradiated rats that had been previously injected with saline following treatment with DES Administration of WR-2721 prior to the irradiation significantly decreased the incidence of mammary tumours to 52.2%. The treatment with DES pellets combined with tamoxifen in the irradiated rats previously injected with saline also markedly suppressed the incidence of mammary tumours even further to 4.4%. Also, the development of mammary tumours was completely prevented in the rats treated with WR-2721 prior to irradiation and then implanted with DES pellets combined with tamoxifen. Conclusions : These results suggest that the administration of WR2721 prior to irradiation has an inhibitory effect on the initiation phase, resulting in a partial reduction of mammary tumour development, and that the combination of WR-2721 at the initiation phase with tamoxifen at the promotion phase is quite effective in preventing mammary tumourigenesis induced by radiation.

Inhibitory effects of WR-2721 and cysteamine on tumor initiation in mammary glands of pregnant rats by radiation.

We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with gamma rays from a (60)Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to gamma rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2. 6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER(+)PgR(+) type. Administration of WR-2721 produced no tumors of the ER(+)PgR(+) type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17beta concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on theinitiation phase during mammary tumorigenesis.

Sparing radiation-induced damage to the physis by radioprotectant drugs: laboratory analysis in a rat model.

The radioprotectant compound amifostine (S-2[3-aminopropylamino]-ethylphosphorothioic acid), administered prior to radiotherapy, has been demonstrated to provide differential protection of normal cells from the damaging effects of ionizing radiation. The aim of this pilot was to determine if amifostine could preserve the integrity of, or minimize the damage to, the physis during exposure to radiation in an animal model. Thirty weanling Sprague-Dawley rats were randomized into five groups of six animals each. Groups 1 and 2 received a single exposure to radiation consisting of 12.5 and 17.5 Gy, respectively. Groups 3 and 4 received similar exposures of 12.5 and 17.5 Gy, respectively, but with prior administration of amifostine at 100 mg/kg. Group 5 (control) received neither radiation nor amifostine. At 6 weeks, femoral and tibial lengths were measured in treated and untreated hindlimbs and compared with the baseline lengths to calculate growth. Concordant with previous reports in the literature, the radiation doses of 12.5 and 17.5 Gy reduced net femoral growth in length by a mean of 23% (range = 12-33%, SD = 7.41) and 59% (range = 54-64%, SD = 4.45), respectively, in the irradiated limb. Amifostine reduced anticipated growth loss normally resulting from a single 12.5-Gy radiation dose by 48.9% in the femur, 13.1% in the tibia, and 27.6% overall in the total limb (p < or = 0.05). Similarly, anticipated growth loss from a single 17.5-Gy radiation dose was reduced by 30.8% in the femur, 20.3% in the tibia, and 25.7% overall in the total limb (p < or = 0.05). Amifostine administered prior to clinically relevant radiation exposures significantly reduced the amount of anticipated growth arrest in our animal model.

Protective effects of the thiophosphate amifostine (WR 2721) and a lazaroid (U83836E) on lipid peroxidation in endothelial cells during hypoxia/reoxygenation

Little is known about pharmacological interventions with thiophosphates or lazaroids in endothelial cells injured by hypoxia/reoxygenation with respect to membrane lipid peroxidation (LPO) caused by reactive oxygen species. Therefore, a cell line of bovine aortic endothelial cells was studied after 120-min hypoxia followed by 30-min reoxygenation, resulting in moderate and predominantly reversible injury (energy depression/cytosolic Ca 2+-accumulation during hypoxia, which almost normalized during reoxygenation; membrane blebs, an increasing amount of lysosomes, vacuolization, lipofuscin formation, alterations in mitochondria size, some lyzed cells). 18.9 ± 4.3% of the cells died. Radical-induced LPO measured as malondialdehyde continuously increased to 2.18 ± 0.17 nmol/mg of protein after reoxygenation vs control (0.41 ± 0.13, P < 0.05). Simultaneously, the content of 4-hydroxynonenal, a novel indicator of LPO, increased from 0.02 ± 0.01 to 0.11 ± 0.02 nmol/mg of protein ( P < 0.01). The results support the assumption that reoxygenation injury is accompanied by an increase in membrane LPO, causing structural and functional disturbances in the monolayer. The thiophosphate WR 2721 [S-2-(3-aminopropylamino) ethylphosphorothioic acid] and the lazaroid U83836E {(-)-2-[[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl] methyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol (dihydrochloride)} were effective scavengers of ·OH, being more efficient than trolox C (6-hydroxy-2,5,7,8-tetramethylchroman-2-carbon acid) used as standard ( ec 50: 12, 5 and 15 μM, respectively, measured by electron spin resonance spectroscopy). One mM WR 2721, 10 μM U83836E, and 5 μM trolox C reduced formation of malondialdehyde during hypoxia/reoxygenation to 53 ± 7, 51 ± 10 and 48 ± 6%, respectively ( P < 0.05 each, versus control). In general, WR 2721 and U83836E prevent radical-induced membrane LPO in a model of endothelial cells injured by hypoxia/reoxygenation. The use of these two agents is a new approach to protect the endothelium against oxidative stress.

Pharmacokinetics of WR-2721 [Amifostine, Ethyol®, S-2-(3 Aminopropylamino)Ethylphosphorothioic Acid] in Patients with Metastatic Ewing's Sarcoma (Pediatric Oncology Group Study) 799

Pharmacokinetics of WR-2721 [Amifostine, Ethyol®, S-2-(3 Aminopropylamino)Ethylphosphorothioic Acid] in Patients with Metastatic Ewing's Sarcoma (Pediatric Oncology Group Study) 799

Repression of c-myc gene expression by the thiol and disulfide forms of the cytoprotector amifostine.

The clinically approved cytoprotector amifostine, designated WR-2721, [S-2-(3-aminopropylamino)ethylphosphorothioic acid], protects against both radiation and drug-induced mutagenesis in animal systems. These effects extend over a wide concentration range making amifostine a strong candidate for evaluation as a possible cancer chemopreventive agent. To better identify and develop potential intermediate biomarkers for chemoprevention at the molecular level we applied the technique of differential display RT-PCR to assess the effects of both the thiol (SH), i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of amifostine on gene expression in CHO-AA8 cells. Cells were exposed to either 40 microM or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in corresponding untreated control cells. One band that showed a differential response was sequenced and was found to have 78% homology with a segment of the human pHL-1 cDNA clone contained in GenBank. This clone contains a COX III mitochondrial DNA insert and two exons of human c-myc. Northern blot analyses were performed by using the cloned human c-myc exon 1 probe to confirm whether c-myc gene expression was affected. Repression of c-myc expression was observed under all of the conditions evaluated. An exposure of cells to 40 microM of the disulfide form of amifostine was the most effective in repressing c-myc, i.e. 27% of control level. A concentration of 4 mM of the disulfide form reduced gene expression to 45% of the control level, while the thiol form was less effective, with 4 mM and 40 microM concentrations reducing c-myc gene expression to 65% and 46% of control levels, respectively.

Comparison of five agents in protecting the cochlea against the ototoxic effects of cisplatin in the hamster

The purpose of this investigation was to study the ameliorating effects of four agents on cisplatin-induced ototoxicity. Hamsters were given a series of five cisplatin injections either alone or in combination with sodium thiosulfate (STS), diethyldihydrothiocarbamate (DDTC), and S-2(3-aminopropylamino) ethylphosphorothioic acid (WR-2721), or fosfomycin. Ototoxicity was assessed anatomically by quantifying the extent of cochlear damage with the scanning electron microscope and physiologically with measures of the auditory brain stem response. When administered alone, cisplatin induced widespread loss of outer hair cells (OHCs) along much of the cochlea in the hamster, especially in the basal and middle turns, with an average survival of only 56% of the OHC population. In contrast, inner hair cells resisted cisplatin ototoxicity in the hamster. Thus the ameliorative effects of the different test agents were assessed by counting the number of surviving OHCs in each treatment group and comparing with cisplatin-treated controls. STS provided the most effective protection against the ototoxic effects of cisplatin, yielding 91% survival of OHCs. DDTC also reduced the ototoxic effects of cisplatin, yielding 68% survival of OHCs. Cotreatment with WR-2721 and fosfomycin yielded 45% and 52% OHC survival, respectively, and thus did not provide any chemoprotection. The results closely paralleled those based on auditory brain stem response recordings in that the magnitude of threshold shift was proportional to the amount of OHC loss; also, the amount of threshold shift at each frequency was in good agreement with the pattern of hair cell loss along the cochlear spiral. Thus both histologic and physiologic results suggest that STS and DDTC hold promise for ameliorating the ototoxic effects of cisplatin chemotherapy. (Otolaryngol Head Neck Surg 1997;117:493-500.)

The inhibition of radiation-induced mutagenesis by the combined effects of selenium and the aminothiol WR-1065

In order to evaluate the anti-mutagenic effects of the potential chemoprotective compounds selenium and ( S)-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-1065), CHO AA8 cells were exposed to both compounds either individually or in combination prior to irradiation. Mutation frequency following exposure to 8 Gy was evaluated by quantitation of the mutations detected at the hprt locus of these cells. Protection against radiation-induced mutation was observed for both 30 nM sodium selenite or 4 mM WR-1065. In addition, the protection against mutation induction provided by the combination of these agents appeared additive. In contrast, sodium selenite did not provide protection against radiation toxicity when provided either alone or in conjunction with WR-1065. In order to evaluate the possible mechanisms of the anti-mutagenic effects observed in these cells, glutathione peroxidase (GPx) activity was evaluated following exposure to the chemopreventative compounds. The addition of sodium selenite to the culture media resulted in a 5-fold increase in GPx activity, which was unaltered by the presence of the WR-1065. Northern analysis of RNA derived from these cells indicated that selenium supplementation resulted in a marginal increase in the mRNA for the cytosolic GPx (GSHPx-1) which was insufficient to account for the stimulation of GPx activity observed in cellular extracts. These results suggest that selenium and WR-1065 offer protection via independent mechanisms and that GPx stimulation remains a possible mechanism of the anti-mutagenic effect of selenium.

Radioprotectors as Late Preventive Agents against Carbon Tetrachloride Induced Liver Necrosis: Protection by 2-(3-Aminopropylamino) Ethylphosphorothioic Acid (WR2721)

Administration of the radioprotective agent 2-(3-aminopropylamino) ethylphosphorothioic acid (WR2721) at 3 or 6 hr after carbon tetrachloride (CCl 4) administration significantly prevented the liver necrosis produced by the hepatotoxin at 24 hr. It is well known that WR2721 does not exert or minimally exerts a protective activity by itself. The compound is activated through dephosphorylation to the free thiol WR1065, a process which is catalyzed by an alkaline phosphatase. We observed that this enzyme was widely distributed in the rat body. The WR2721 pretreatment 30 min before CCl 4 administration modified the CCl 4 levels reaching the liver at 1 hr of poisoning and exerted a significant increase in the covalent binding (CB) of 14CCl 4-reactive metabolites to microsomal lipids at 3 hr. WR2721 did not modify the intensity of the CCl 4-induced lipid peroxidation (LP) process at 1 or 3 hr of poisoning. CCl 4--induced fat accumulation was not prevented when WR2721 was given 6 hr after CCl 4. In fact, protection might be due to a favorable modulation of late events occurring after CB or LP, events that remain to be elucidated.

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, l) trans-1-1,2-diaminocyclohexanetetrachloroplatinum( IV), Pt(dach)Cl 4, (rmformerly called tetraplatin)]. Those data suggested that a direct interaction between WR-2721 and ormaplatin and/or the corresponding Pt(II) drug, Pt(dach)Cl 2, 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 °. 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.

Induction of micronucleated erythrocytes by MEA, AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.

Acute effects of irradiation on the rat brain: protection by glutamate blockade

Riluzole (2-amino-6-trifluorothethoxy benzothiazole), dizocilpine (MK-801; (+)-5-methyl-10,11-dihydro-5 H-dibenzo( a,d)-cyclohepten-5,10-imine maleate), and lamotrigine (3,5-diamino-6-(2,3-dichlorophenyl)-1,2-triazine), agents reported to have neuroprotective actions, and WR2721 ( S-2-(3-aminopropylamino)-ethylphosphorothioic acid), a radioprotector, were evaluated in 15-day-old rats that underwent a 2.5 Gray (Gy) irradiation from a cobalt 60 source. 20 min after irradiation, riluzole (0.5–8 mg/kg), dizocilpine (0.1–1 mg/kg), lamotrigine (25 mg/kg), WR 2721 (75 mg/kg) or vehicle, were injected intraperitoneally. 6 h after irradiation, behavioural and histological evaluations revealed that exposure to 2.5 Gy caused hypolocomotion, stumbling gait and somnolence, which was significantly reduced, from the dose of 4 mg/kg i.p. of riluzole. A dose-dependent protection of neurones in the dentate gyrus, starting from the dose of 1 mg/kg i.p. was also seen. Dizocilpine caused behavioural modifications but significantly reduced neuronal damage. Lamotrigine significantly increased neuronal damage while WR 2721 conferred no protection. In conclusion, two blockers of glutamatergic neurotransmission conferred significant protection against brain damage caused by ionizing irradiation when administered subsequent to exposure.