Modification by ozone of lung tumor development in mice

Mice, either strain A/J or Swiss Webster, were exposed for 18 weeks either to filtered air or to 0.4 or 0.8 ppm ozone for 8 hours daily. Subgroups in each test group received a single ip injection of 1,000 mg urethan/kg or 0.9% sodium chloride vehicle 1 day prior to initiation of the exposure regimen. Tumor incidence in Swiss Webster mice was 0-3% in groups not receiving urethan and was 61-74% in groups receiving urethan. In A/J mice, the corresponding values were 9-38% and 100%, respectively. Exposure to ozone caused a decrease in the number of tumors per lung in urethan-treated mice of both strains, in a dose-dependent manner. There seemed to be a specific decrease in tumors derived from alveolar type II cells in the A/J mice given urethan plus ozone. Most interesting, perhaps, was a significant increase in the number of tumors per lung in A/J mice exposed to 0.8 ppm ozone without urethan, confirming a previous report by others. The corresponding ozone effect on lung tumor development was not observed in Swiss Webster mice.     

The effect of hypobaric hypoxia on misonidazole binding in normal and tumour-bearing mice

The effect of hypobaric hypoxia on the in vivo binding of misonidazole was investigated in normal mice and mice bearing T50/80 or CA NT mammary carcinomas. After the intraperitoneal injection of radiolabelled misonidazole, mice were randomised to breathe either room air or air at 0.5 atmospheres. The distribution of misonidazole in liver, kidney, heart, spleen and tumour tissue, 24 h later, was studied by scintillation counting and by autoradiography. Significantly higher misonidazole binding occurred in the livers (x2.5), kidneys (x2.4), spleens (x2.9) and hearts (x1.8) of hypoxic mice compared to controls. Hypobaric hypoxia was associated with a greater than four-fold increase in misonidazole binding within T50/80 tumours. However, significantly higher binding was not demonstrated within CA NT tumours after exposure of tumour-bearing animals to hypoxic conditions. In autoradiographs of hypoxic liver, labelling was intense in regions near to hepatic veins but sparse in areas surrounding portal tracts. This pattern was striking and consistent. In hypoxic kidney, labelling was most intense over tubular cells, less intense over glomeruli and sparse in the renal medulla. It is likely that the hepatic and renal cortical distributions of misonidazole binding reflect local oxygen gradients.     

Partial characterization of an antigen in spontaneous murine mammary tumors

Mammary carcinomas of three strains of mouse (Swiss Webster, A, and C3H/He) were investigated for the presence of a tumor-specific antigen. The antigen could be detected only in the outbred Swiss Webster mice. When rabbit antisera to pools of mammary tumor and normal tissues were absorbed three or more times with normal tissues (heart, lungs, spleen and liver) and examined by gel immunodiffusion, the results indicated that the antigen was present in tumor tissue but not in normal tissue. The antigen could not be demonstrated in other organs of tumor-bearing mice. Moreover, the precipitin band indicative of tumor antigen was easily removed by absorbing antitumor sera with pooled mammary tumor tissues. The results of lipid extraction procedures suggested that the antigen was not a lipid. The fact that it was not inactivated at 56°C for at least 30 minutes suggested that the antigen was fairly heat-stable. Trypsinization destroyed the ability of the antigen to react with absorbed anti-tumor sera, suggesting that it was probably protein in nature. With the cellular fractionation procedures employed, the antigen remained in the supernatant of the 105,000 × g fraction. This suggested that it was a cytoplasmic constituent. The results of immunofluorescent studies substantiated the impression that the antigen was in the cytoplasm of the tumor cells. No membrane or nuclear fluorescence could be detected.     

Dietary restriction, cell proliferation and carcinogenesis: a preliminary study

Four groups of female Swiss Webster mice were given either laboratory chow or a purified (semi-synthetic) diet (AIN-76A) either ad libitum or at 75% of the ad libitum rate for about 30 days. Three tissues, the crypt cells of the jejunum, the dermis and the basal epithelial cells of the esophagus were investigated using [3H]thymidine labelling and by counting mitoses; four other tissues, the alveolar cells of the mammary gland, the crypt cells of the duodenum and colo-rectum, and the transitional cells of the urinary bladder were examined using [3H]thymidine labelling only. In each case dietary restriction led to a reduction of cellular proliferation assessed by these indices. The potential of the approach for the study of the effects of dietary modification on the induction of cancer is discussed.     

Binding to cellular macromolecules as a possible mechanism for the cytotoxicity of misonidazole

Reduction of the nitro group occurred when [14C]misonidazole was treated with zinc dust in aqueous solution in the presence of ammonium chloride. When the reduction mixture was allowed to react with calf thymus DNA or bovine albumin, radioactivity was bound to both DNA and protein. Under the same conditions, misonidazole did not bind to these macromolecules. Analysis of the reduction mixture indicated that the hydroxylamine, amine, and hydrazo derivatives of mizonidazole were the major products. In a number of tissues of C3H mice after administration of [14C]misonidazole, radioactivity was detected in the DNA, RNA, and protein fractions. Similar results were also obtained with Chinese hamster ovary cells incubated with the drug in the absence of oxygen. It is postulated that nitroreduction and binding of the nitroreduction products to macromolecules is a probable mechanism for the mutagenic and cytotoxic properties of misonidazole.     

Effect of acute and chronic misonidazole administration on peripheral-nerve electrophysiology in mice.

I.p. administration at several dose levels over periods of up to 12 weeks, or continuous i.v. infusion of high doses of misonidazole (MISO) for 15 h, produced no significant change in peripheral nerve conduction velocity (NCV) and did not prevent the normal increase in NCV as the animals matured from 12 to 24 weeks of age. Peripheral NCV (sural nerve) was reduced in both MISO-treated and control mice with hind-limb tumour implants, presumably owing to physical pressure due to tumour growth. In addition, neither the medial nerves nor the tibial nerve in the normal limbs of the tumour-implanted, drug-treated animals showed any change. Consequently our earlier and present studies do not confirm the recent reports of changes in NCV following either acute or chronic MISO administration to mice.     

Metabolism and excretion of [3H]misonidazole by hypoxic rat liver

Our investigation was initiated to determine if misonidazole's biological activity is related to hypoxia-dependent, reductive biotransformation to form reactive metabolites. This study was facilitated by the synthesis of [3H]misonidazole and by use of the isolated perfused rat liver as a model system for hypoxic tissue. The perfused rat liver was verified as an appropriate model system to study misonidazole (MISO) metabolism. This was done by demonstrating that the perfused liver produced the same metabolites as those isolated from rats given MISO, albeit reductive metabolism was much less in rats. Reductive metabolism of MISO by perfused livers was enhanced (estimated by measuring the rate of 1-[2-aminoimidazol-1-yl]-3-methoxy-2-propanol production) by hypoxic conditions. Formation of a MISO-derived glutathione conjugate (MISO-GSH) and covalent binding of MISO-derived radioactivity to tissue protein was also enhanced by hypoxia. Depletion of hepatic GSH with diethyl maleate increased the extent of covalent binding to protein under both aerobic and hypoxic conditions, and greatly diminished the formation of MISO-GSH. These results support the hypothesis that hypoxic conditions facilitate reductive metabolism of MISO to an alkylating agent, and that GSH plays an intervening role in the alkylation reaction.     

Distribution of pimonidazole and RSU 1069 in tumour and normal tissues

The tritium-labelled analogues of pimonidazole and RSU 1069 were injected into mice bearing the KHT murine sarcoma which has a hypoxic cell fraction of approximately 10%. The distribution of activity at 24 h was recorded using autoradiography and measurement of tissue activity. Autoradiographs with both drugs showed high activity in particular cells within tumour, eye (melanin-associated cells), eyelid (Meibomian gland), liver (centrilobular area), skin (sebaceous gland and melanin), stomach (squamous area), footpad, oesophagus, labial gland, Zymbal's gland, preputial gland, parotid gland (intralobular ducts) and airway epithelium. These tissues had previously been identified as sites of binding of misonidazole. The measurement of total tissue radioactivity showed significantly higher activity in liver, eyelid (Meibomian gland), oesophageal lining, kidney and labial gland than was found in the tumour.     

Diversity of the promoting action of cyclosporine on the induction of murine lymphoid tumors

We have previously demonstrated that cylosporine (CsA), a powerfulimmunosuppressant, enhanced the development of thymic lymphomasin Swiss Webster mice initiated with a single subcarcinogenicdose of N-methyi-N-nitrosourea and enhanced the spontaneousdevelopment of thymic tumors in AKR mice. In the present study,we examined whether the initiation of mice with a single doseof -radiation modified the target cell specificity of the CsApromotion of lymphoma induction. Male Swas Webster and C57B/6mice were divided into four groups. The mice in group 1 and2 of both strains were given a single dose (350 rad) of --radiation;10 days thereafter group 1 was given a basal diet and group2 a basal diet containing 0.015% CsA for 25–35 weeks.Groups 3 and 4 consisted of control mice without radiation andwere maintained on a basal or a CsA diet. None of the mice ineither strain in groups 1, 3 and 4 developed tumors. Eighteenout of 39 (46%) Swiss Webster mice that received radiation followedby a CsA diet developed tumors involving mesenteric lymph nodesand the spleen. The tumor cells had immunoglobulins on theirsurface and were negative for Thy 1, suggesting lymphomas ofB cell lineage. Four mice (10%) developed thymic tumors whichwere positive for Thy 1. Seven of 14 (50%) C57B/6 treated withradiation and CsA developed thymic tumors, while none developedtumors of B cell lineage. The results indicate that CsA is apotent promoter of the induction of lymphomas in mice and thatthe cell type is determined by the type of initiating agentsand the strain of mice.     

Peripheral electrophysiological parameters in mice treated with misonidazole.

The clinical use of the radiosensitizer misonidazole may be limited by the incidence of peripheral neuropathy reported following total doses in excess of 18 g. A recent report noted a decrease in nerve conduction velocity following a single i.p. injection of 1 mg/g misonidazole in mice. The present study was unable to confirm such changes when nerve conduction velocity measurements were made in situ or in isolated sural, tibial or median nerves of mice. Other electrophysiological parameters such as threshold, strength-duration curves, refractory time or the ability to carry high-frequency stimulation also showed no change. However, it was noted that a single administration of the radio-sensitizer produced a marked decrease in body temperature which persisted for at least 2 h after the elimination of the drug from the blood serum. The physiological response of reduction of body temperature may protect the mouse against the effect of the toxic chemical species involved in the induction of neurotoxicity.     

Ethionine carcinogenesis in CD-1, BALB/c and C3H mice

In two separate in vivo studies, ethionine was evaluated forcarcinogenic activity in mice. In the first study, DL-ethioninewas fed in a chow diet at 0 (controls), 0.1 (low dose, LD) and0.25% (high dose, HD) concentrations to the following groupsof mice (30 animals/group): Swiss Webster CD-I females, BALB/cmales, and C3H/HeN males and females. Because of severe toxicity,BALB/c females were fed 0.05% (LD) and 0.1% (HD) ethionine.The Swiss and BALB/c mice were maintained on their respectivediets for up to 105 weeks before killing whereas the C3H micewere killed at 68 weeks because of the high spontaneous incidenceof liver tumors hi this strain. Hie percentages of animals atrisk (surviving the time to the first liver tumor recorded ineach sex and strain) that bore liver tumors were as follows:Swiss female control, 0% (0/29), Swiss female LD, 87% (20/23);Swiss female HD, 89% (16/18); C3H male controls, 35% (8/23);C3H male LD, 55% (16/29); C3H male HD, 58% (15/26); C3H femalecontrols, 5% (1/20); C3H female LD, 60% (12/20); C3H femaleHD, 92% (12/13); BALB/c male controls, 4% (1/23); BALB/c maleLD, 8% (2/24); BALB/c male HD, 31% (5/16); BALB/c female controls,0% (0/30); BALB/c female LD, 52% (14/27); and BALB/c femaleHD, 92% (12/13). The female mice were more responsive than themales hi developing liver tumors. The results of the feedingstudy are compared with those obtained in a second study hiwhich C3H female mice were in-tubated with 0, 150 or 500 mgDL-ethionine/kg body wt three times per week for 30 weeks andkilled at 2 years. Only the LD mice showed a significantly increasedincidence of liver tumors (20/39) as compared to controls (12/41)or HD mice (7/37) hi the latter study. The hepatic levels ofthe major ethionine metabolite and methylase inhibitor, S-adenosyl-ethionine(AdoEt), as well as of the endogenous methyl group donor, S-adenosylmethionine(AdoMet) were determined in Swiss female mice fed either 0.1or 0.3% in the diet for 1–6 weeks. Hepatic AdoEt levelsranged from 37 to 80 /ig/g h'ver hi the LD animals and from61 to 203 /ig/g liver in the HD group; levels of the endogenousmetabolite AdoMet correspondingly dropped to 65% of the normallevels. The present results (0 extend to different strains andto both sexes previous observations demonstrating the hepatocarcinogenkactivity of ethionine hi mice; and (ii) indicate that as hithe rat such activity may be exerted through the formation ofAdoEt.     

Distribution and tumor penetration properties of a radiosensitizer 2-[14C] misonidazole (Ro 07-0582), in mice and rats as studied by whole-body autoradiography

Summary The hypoxic cell radiosensitizer 2-[¹⁴C] misonidazole: 1-(2-nitro-1-imidazolyl)-3-methoxy-2-propanol (Ro 07-0582) MISO was administered to mice, control rats, and rats bearing chemically induced rhadbdomyosarcoma. The dose injected was 250 mg/kg and the delivered activity was 100 Ci/kg. Whole-body autoradiography was performed in all animals. We noted the highest uptake of radioactivity in the liver and the kidney. In the liver there was an accumulation of [¹⁴C] from 5 min to the 2 hour after treatment, followed by a decrease; this observation is probably related to the metabolic pathway of the drug. The radioactivity was also concentrated in the renal medulla (30 min after injection); this organ is the excretion route for most of the misonidazole or its metabolites. Fecal excretion is also important following biliary elimination. Radioactivity is present in the central nervous system in the first hours after dosage. [¹⁴C] Tumor activity was lowest 5 min after IP treatment. By contrast, 12 h after administration of labeled compound the highest activity was detected in this tissue.The work described in this paper was supported by a grant from the Fédération Nationale des Centres de Lutte Contre le Cancer (France)     

Evoked potentials in rats with misonidazole neurotoxicity

Summary Spinal and cortical somatosensory potentials were recorded weekly in rats treated with misonidazole at a dose of 300 mg/ kg/ day until clinical signs of severe neurotoxicity developed. After sacrifice, the brains, spinal cords and sciatic nerves were examined. All treated rats developed necrotic brain stem lesions identical to those previously described by Griffin and by us. Two rats had mild demyelination of spinal cord white matter tracts, but only one of these rats had changes in the SEPs. Interestingly, changes in the cervical and cortical SEP from this rat were only transient.We conclude that misonidazole neurotoxicity in the rat predominantly affects the brain stem nuclei adjacent to the fourth ventricle without involving the lemniscal pathways that are situated ventromedially in the brain stem. Therefore, somatosensory evoked potentials (SEPs) are not useful for the diagnosis of MISO neurotoxicity.     

Increased therapeutic benefit through the addition of misonidazole to a nitrosourea-radiation combination

The potential therapeutic advantage of including the radiosensitizer misonidazole (MISO) in a treatment regimen combining systemic chemotherapy and localized radiotherapy was assessed in the KHT sarcoma. Tumor-bearing C3H mice were treated with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, administered either alone or in combination with MISO, 24 h prior to irradiating the tumors with doses ranging from 0-35 Gy. The sensitizer exposure was either a single dose of 2.5 mmol/kg or multiple injections given every 0.5 h for 8 h to maintain a sensitizer blood level of approximately 100 micrograms/ml. The chemotherapeutic agent was administered simultaneously with the single sensitizer dose or 3 h into the chronic sensitizer dosing schedule. Tumor responses to the combined modality treatments was determined using end points of tumor regrowth delay and animal tumor-free survival. Skin reactions in the treatment field were scored as a measure of normal tissue complications. MISO, administered as a single dose in the combined modality therapy, enhanced the tumor response by a factor of approximately 1.8-1.9 while increasing skin reactions approximately 1.1- to 1.2-fold. Combining 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea with chronic MISO administration prior to irradiation increased the resultant tumor control probability 1.5- to 1.8-fold without enhancing normal tissue complications. Consequently using MISO as a chemopotentiator in a 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea-radiation combination can yield a significant therapeutic benefit.     

Misonidazole neurotoxicity in the mouse: evaluation of functional, pharmacokinetic, electrophysiologic and morphologic parameters

The neurotoxic effects of chronic administration of misonidazole (0.3 mg/g/day, 5 times weekly) were investigated in Balb/cKa mice over 12 weeks; a variety of measurements were used, including functional and clinical performance, morphologic, electrophysiologic and pharmacokinetic parameters. The half life of drug for a single dose was greater in brain (3 hrs) compared to serum (1.2 hrs); these values decreased to 1.9 hrs and 1.0 hrs respectively after 3 weeks. Misonidazole induced a peripheral lesion after three weeks with a total administered dose of 13.5 g/m2 or exposure dose of 57 to 75 mM X hrs, which is similar to the doses that cause neuropathy in humans. There was some suggestion of a central neurological deficit related to locomotor control and balance; however, no gross morphological damage was found in the brain. The sequence of effects demonstrated began at 3–4 weeks and included: 1) morphologic damage to peripheral nerves; 2) hyperactivity and listlessness; 3) a decrease in rotarod retention time which reached a value 50% of that of saline injected control mice at 8–10 weeks; 4) walking on tip-toes with a slightly hunched back (4–6 weeks); and 5) an increase in hind foot splay (6–7 weeks). The morphologic damage primarily involved the more distal portions of the nerves supplying the interosseous muscles and footpads of the hind limbs. The damage was more severe and progressed more rapidly with time in these distal areas compared to the more proximal regions of the nerves. No marked changes were found in nerve conduction velocity although neuropathy produced by acrylamide produced significant decreases. The changes in neurological parameters reported here may be useful in the further evaluation of hypoxic cell radiosensitizers.     

In vitro assessment of the oncogenic potential of nitroimidazole radiosensitizers

Two hypoxic cell radiosensitizers, RSU-1069 and Ro-03-8799 were investigated for their in vitro cytotoxicity and ability to induce oncogenic transformation and sister chromatid exchanges in the C3H 10T1/2 cell system. Their effects were then compared to those of the clinically used sensitizer misonidazole. Equitoxic doses of Ro-03-8799 and RSU-1069 were approximately 3-fold and 150-fold less than misonidazole, respectively, with both agents exhibiting dose and contact time dependence for cell killing. Both sensitizers appeared no more oncogenic than misonidazole when administered at equitoxic dosages. At doses of equivalent sensitizing efficiencies relative to misonidazole, RSU-1069, but not Ro-03-8799, induced significantly higher transformation incidence. In conjunction with gamma-irradiation, both Ro-03-8799 and misonidazole induced an additive transformation response. Preliminary studies also indicate that RSU-1069, at a concentration of 0.03 mM, induced significantly higher sister chromatid exchanges (SCE) per chromosome than either Ro-03-8799 or misonidazole at concentrations 30-fold higher. Although several earlier studies have indicated that RSU-1069 may be more efficient than misonidazole as an hypoxic cell sensitizer, the present findings suggest that it may also carry a higher risk of inducing tumors by itself at clinically relevant concentrations.     

A biochemical method for assessing the neurotoxic effects of misonidazole in the rat.

A proven biochemical method for assessing chemically induced neurotoxicity has been applied to the study of the toxic effects of misonidazole (MISO) in the rat. This involves the fluorimetric measurement of beta-glucuronidase and beta-galactosidase activities in homogenates of rat nervous tissue. The tissues analysed were sciatic/posterior tibial nerve (SPTN) cut into 4 sections, trigeminal ganglia and cerebellum. MISO administered i.p. to Wistar rats in doses greater than 300 mg/kg/day for 7 consecutive days produced maximal increases in both beta-glucuronidase and beta-galactosidase activities in th SPTN at 4 weeks (140-180% of control values). The highest increases were associated with the most distal secretion of the nerve. Significant enzyme-activity changes were also found in the trigeminal ganglia and cerebellum of MISO-dosed rats. The greatest activity occurred 4-5 weeks after dosing, and was dose-related. It is concluded that, in the rat, MISO can produce biochemical changes consistent with a dying-back peripheral neuropathy, and biochemical changes suggestive of cerebellar damage. This biochemical approach would appear to offer a convenient quantitative method for the detection of neurotoxic effects of other potential radio-sensitizing drugs.     

KIH-802, an acetohydroxamic acid derivative of 2-nitroimidazole,as a new potent hypoxic cell radiosensitizer: radiosensitizing activity,acute toxicity,and pharmacokinetics

Summary The radiosensitizing activity, acute toxicity, and pharmacokinetics of a new hypoxic cell radiosensitizer, potassium 2-nitroimidazole-1-acetohydroxamate (KIH-802), were compared with those of misonidazole (MISO) and etanidazole (SR-2508). The radiosensitizing activity of KIH-802 was slightly higher than that of MISO and SR-2508 in vitro and was similar to or slightly higher than that of MISO or SR-2508 in vivo. The acute toxicity of KIH-802 was slightly higher than that of MISO. The concentrations of KIH-802 in the brains and peripheral nerves of mice were as low as those of SR-2508 and lower than those of MISO. This work was supported in part by Grants-in-Aid from the Japanese Ministry of Education, Science and Culture (62010070, 63010070)     

Kinetics of depression and recovery of murine hepatic cytochrome P-450 levels after treatment with the interferon inducer polyriboinosinic-polyribocytidylic acid

In vivo treatment of randombred Swiss Webster mice with polyriboinosinic-polyribocytidylic acid (poly l X poly C) inhibited the induction of cytochrome P-450's by both 3-methylcholanthrene [(MCA) CAS: 56-49-5] and phenobarbitol [(PB) CAS: 50-06-6]. Concomitant treatments with poly l X poly C and a single dose of MCA inhibited the induction of P-450's for 24 hours and delayed the obtainment of the MCA-induced P-450 levels for approximately 48-72 hours. When cytochrome P-450 levels were induced by four successive daily treatments of MCA or PB and when poly l X poly C was given on only the 1st day, induction of P-450's was completely suppressed for 24 hours and obtainment of the maximal P-450 level was delayed by 72-96 hours. Treatment with poly l X poly C of animals preinduced for P-450's by four successive daily treatments with either PB or MCA decreased the P-450 content to the noninduced basal level within 24 hours. The effect was temporary in the MCA-treated mice since P-450 content recovered to the MCA-preinduced levels within 72 hours. PB-dependent P-450 induction was short lived, and no recovery occurred after poly l X poly C treatment of PB-preinduced mice. Reduced hepatic cytochrome P-450 contents correlated with decreased abilities of liver homogenates to metabolically activate benzo [a]pyrene (CAS: 50-32-8) and N-acetyl-2-aminofluorene (CAS: 53-96-3), as scored in an Ames Salmonella typhimurium revertant assay.     

Response of murine tumours to combinations of CCNU with misonidazole and other radiation sensitizers

The effect of combinations of the conventional chemotherapeutic agent 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and nitroimidazole radiation sensitizers was evaluated in female C3H mice. Tumour response to single-agent or combination therapy was assessed in a tumour growth-delay assay. In the KHT sarcoma the simultaneous addition of misonidazole (MISO) was found to increase significantly the tumour growth delay resulting from CCNU treatment. The observed enhancement ratios (ER) increased with MISO dose, and ranged from 1.3 to 1.9 for sensitizer doses of 0.25-1.0 mg/g. The combination of CCNU and 1.0 or 0.5 mg/g MISO in the RIF-1 tumour or the MT-1 tumour produced ERs of approximately 2.0 and approximately 1.5 respectively. In the KHT sarcoma a series of other nitroimidazole sensitizers, including Ro-05-9963, SR-2555, SR-2508 and metronidazole (METRO), were also evaluated at equimolar doses (5 mmol/kg) in combination with a 20mg/kg dose of CCNU. Unlike MISO, these compounds in general failed to enhance the CCNU cytotoxicity in this tumour model. However, SR-2508 did enhance the response of the RIF-1 tumour to large single doses of CCNU, though not as much as MISO. Normal-tissue toxicity was determined using peripheral white blood cell (WBC) counts 3 days after treatment. CCNU doses of 10-50 mg/kg given either alone or in simultaneous combination with 0.5 or 1.0 mg/g MISO were studied. WBC toxicity increased with CCNU dose, but the addition of MISO at either dose did not significantly enhance this normal-tissue toxicity.