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Antibiotics are often applied in aquaculture to prevent fish diseases. These substances can cause disturbances on receiving waters, when not properly eliminated from the aquaculture effluents. In this work, ozone (O 3 ) was investigated as a possible oxidizing agent to remove fishery antibiotics from aquaculture effluents: florfenicol (FF), oxytetracycline (OTC), sulfadimethoxine (SDM), sulfamethoxazole (SMX), and trimethoprim (TMP). Batch experiments were performed using ultrapure water and aquaculture effluents spiked with a mixture of target antibiotics at relatively high concentrations (10 mg L -1 each). OTC, SMX and TMP were fully removed (< 30 min) regardless of the tested conditions, mainly by O 3 direct attack. In contrast, FF was partially removed in 30 min (∼ 10 and 60%, in aquaculture effluents and ultrapure water, respectively), but only in the presence of hydroxyl radicals (HO • ), the FF concentrations reaching levels below the detection limits in ultrapure water after 60 min. In the case of SDM, its degradation was highly influenced by the selected water matrix, but with removals always higher than 68%. In continuous-flow experiments applying more environmentally relevant antibiotic concentrations (100 ng L -1 each) and low O 3 doses (1.5 mg L - 1 ), ozonation highly removed (> 98%) all tested antibiotics from aquaculture effluents with a hydraulic retention time (HRT) of 10 min, except FF (68%). Although by-products were detected in treated samples, zebrafish (Danio rerio) embryotoxicity tests did not show a toxicity increase by applying this ozonation treatment. Ozonation is thus a possible solution to remove antibiotics from aquaculture effluents. Still, full-scale studies in aquaculture farms are needed, and generation of HO • may be favoured to readily oxidize the FF antibiotic., (Copyright © 2022. Published by Elsevier Ltd.)

Sulfonamides are the second most widely used group of veterinary antibiotics which are often detected in the environment. They are eliminated from freshwaters mainly through photochemical degradation. The toxicity of sulfadimethoxine (SDM) was evaluated with the use of Lemna minor before and after 1- and 4-h irradiation in a SunTest CPS+ solar simulator. Eight endpoints consisting of: number and total area of fronds, fresh weight, chlorophylls a and b, carotenoids, activity of catalase and guaiacol peroxidase, and protein content were determined. The total frond area and chlorophyll b content were the most sensitive endpoints with EC50 of 478 and 554 μg  L -1 , respectively. The activity of guaiacol peroxidase and catalase increased at SDM concentrations higher than 125 and 500 μg  L -1 , respectively. The SDM photodegradation rate for first order kinetics and the half-life were 0.259 h -1 and 2.67  h, respectively. The results show that the toxicity of irradiated solutions was caused by SDM only, and the photoproducts appeared to be either non-toxic or much less toxic to L. minor than the parent compound. To study the recovery potential of L. minor, after 7 days exposure in SDM solutions, the plants were transferred to fresh medium and incubated for the next 7 days. L. minor has the ability to regenerate, but a 7-day recovery phase is not sufficient for it to return to an optimal physiological state.

Background: Free bilirubin (Bf), the unbound fraction of unconjugated bilirubin (UCB), can induce neurotoxicity, including impairment of the auditory system, which can be assessed by brainstem auditory evoked potentials (BAEPs). We hypothesized that albumin might reduce the risk of neurotoxicity by decreasing Bf and its translocation into the brain., Aim: To determine the effects of albumin on BAEPs and brain bilirubin content in two Gunn rat pup models of acute hyperbilirubinemia., Methods: We used Gunn rat pups, which have a deficiency of the bilirubin-conjugating enzyme UGT1A1. We induced haemolysis by injection of phenylhydrazine (phz) into 14-days old pups. Subsequently, pups were treated with either i.p. human serum albumin (HSA; 2.5 g/kg; n = 8) or saline (control, n = 8). We induced acute neurotoxicity by injecting 16-days old pups with sulphadimethoxine (sulpha) and treated them with either HSA (n = 9) or saline (control, n = 10). To assess bilirubin neurotoxicity, we used the validated BAEP method and compared relevant parameters; i.e. peak latency values and interwave interval (IWI) between peak I and peak II, a marker of acute neurotoxicity., Results: Phz and sulpha significantly increased IWI I-II by 26% and 29% (P < 0.05) in the haemolysis and the displacement model, respectively. Albumin completely prevented the increase of IWI I-II in either model. The beneficial effect of albumin in the displacement-model by means of normal BAEPs was in line with less bilirubin in the brain (NS). Interestingly, in the haemolysis model the accumulation of total bilirubin in the brain was unaltered, and BAEPs still appeared normal. This might advocate for a role of brain Bf which was calculated and showed that albumin treatment non-significantly reduces Bf concentrations in brain, compared with saline treatment., Conclusions: Albumin treatment is neuroprotective in acute hyperbilirubinemia in Gunn rat pups. Our present results underline the importance of functional diagnostic test of neurotoxicity above biochemical concentrations., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The application of manure to fertilize arable lands is one of the major means through which veterinary sulfonamides (SAs) enter the environment. Little is known about the capacity of woody plants to phytoremediate this class of antibiotics. To this purpose we performed preliminary studies to evaluate Salix fragilis L. response to sulfadimethoxine (SDM) by investigating both its ability to absorb and tolerate doses of SDM found in fresh faeces of treated calves. Forty cuttings were exposed to either 0, 0.5, 1, or 2 mM of SDM for one month. Decreases in photosynthetic electron transport rate and net CO2 assimilation after 25 days for the higher SDM concentrations were noticed. Moreover, alterations in root morphology of treated plants were observed and further investigated through electron microscopy. However, collected data revealed high root accumulation potential. These preliminary results are promising as they demonstrate that Salix fragilis L. can both absorb and tolerate high concentrations of SAs.

We have shown phosphoinositide 3-kinase (PI3K)/Akt signaling activation in thyroid capsular invasive carcinomas (CICs), which are highly induced by promotion with sulfadimethoxine (SDM) in a rat 2-stage thyroid carcinogenesis model. To examine the potency of calcitriol, a synthetic vitamin D3 analog, on the development or progression of CICs, male F344 rats were injected with calcitriol (0.1 µg/kg body weight) three times a week intraperitoneally, during an entire period of SDM-promotion for 13 weeks (Experiment 1) or during the last 2 weeks of a 15-week SDM-promotion (Experiment 2). Initiation with N-bis(2-hydroxypropyl)nitrosamine preceded all treatments. In Experiment 1, long-term calcitriol treatment reduced the multiplicity of CICs, while cell proliferation activity, estimated by Ki-67 cell index in the induced CICs, was unchanged with SDM-promotion alone. Considering the strong dependency of promotion with SDM during the early stages on thyroid-stimulating hormone, the reduced multiplicity in Experiment 1 may be due to the effect on an early stage of neoplastic proliferation. Although the magnitude was mild, cell proliferation activity was decreased in existing CICs after short-term calcitriol treatment in Experiment 2, which was associated with a mild decrease in cyclin-dependent kinase-2-positive cells, cytoplasmic immunolocalization of phosphorylated, inactive, Rb protein and a mild increase in nucleocytoplasmic expression of p27(kip1). Although the effect was mild at the late stage of SDM-promotion in this hypothyroidism-related thyroid carcinogenesis model, our results suggest that calcitriol targets cell proliferation via inhibition of a molecular cascade downstream of PI3K/Akt signaling, controlling G1/S transition.

Purpose: Rat thyroid follicular cell carcinomas invading into the thyroid capsule are highly produced by promotion with sulfadimethoxine (SDM) in a rat two-stage thyroid carcinogenesis model. In this study, we investigated the participation of phosphoinositide 3-kinase (PI3K) signaling pathway that is associated with malignant phenotypes of many cancers on the development of SDM-induced capsular invasive carcinomas., Methods: Thyroid proliferative lesions developed 10 or 15 weeks after promotion with SDM in male F344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine were immunohistochemically analyzed with regard to cellular distribution of phosphatase and tensin homolog (PTEN) and Akt isoforms, as well as their downstream molecules., Results: Increased expression of PI3K signaling molecules was evident in association with the development of lesion stages from the early focal hyperplasia to the late carcinomas. Capsular carcinomas, and the less frequent parenchymal carcinomas, exclusively expressed phosphorylated, inactive PTEN, and active Akt isoforms, as did their downstream molecules. Among the Akt isoforms, enhanced expression of Akt1 was more prominent than that of Akt2 in both capsular and parenchymal carcinomas., Conclusions: Activation of the PI3K pathway through phosphorylation of PTEN promotes the high production of capsular carcinomas as well as the development of less frequent parenchymal carcinomas.

During feed preparation at feed mills or during feed mixing in bins at farms, the accidental contamination of feed at trace levels by veterinary drug residues, commonly known as carry-over, can accidentally but frequently occur. To evaluate the concentrations of residual antimicrobials in poultry edible tissues, due to contaminated feed, sulfadimethoxine and doxycycline were administered for 10 days to chickens in poultry feed incurred at the contamination levels frequently found during national feed monitoring programmes (1-5 mg kg(-1)). Sulfadimethoxine and doxycycline residual concentrations detected in muscle (

There is a well-established link between hyperbilirubinaemia and hearing loss in paediatrics, but the cellular mechanisms have not been elucidated. Here we used the Gunn rat model of hyperbilirubinaemia to investigate bilirubin-induced hearing loss. In vivo auditory brainstem responses revealed that Gunn rats have severe auditory deficits within 18 h of exposure to high bilirubin levels. Using an in vitro preparation of the auditory brainstem from these rats, extracellular multi-electrode array recording from the medial nucleus of the trapezoid body (MNTB) showed longer latency and decreased amplitude of evoked field potentials following bilirubin exposure, suggestive of transmission failure at this synaptic relay. Whole-cell patch-clamp recordings confirmed that the electrophysiological properties of the postsynaptic MNTB neurons were unaffected by bilirubin, with no change in action potential waveforms or current-voltage relationships. However, stimulation of the trapezoid body was unable to elicit large calyceal EPSCs in MNTB neurons of hyperbilirubinaemic rats, indicative of damage at a presynaptic site. Multi-photon imaging of anterograde-labelled calyceal projections revealed axonal staining and presynaptic profiles around MNTB principal neuron somata. Following induction of hyperbilirubinaemia the giant synapses were largely destroyed. Electron microscopy confirmed loss of presynaptic calyceal terminals and supported the electrophysiological evidence for healthy postsynaptic neurons. MNTB neurons express high levels of neuronal nitric oxide synthase (nNOS). Nitric oxide has been implicated in mechanisms of bilirubin toxicity elsewhere in the brain, and antagonism of nNOS by 7-nitroindazole protected hearing during bilirubin exposure. We conclude that bilirubin-induced deafness is caused by degeneration of excitatory synaptic terminals in the auditory brainstem.

Acrylamide (AA) has been reported to be formed in fried and baked foods with various concentrations, and exposure levels to AA from cooked foods in children are estimated to be higher than those in adults. In order to evaluate the carcinogenicity of AA exposure during childhood, we conducted a medium-term carcinogenicity study with prepubertal administration of AA followed by treatments of a multi-organ-targeted genotoxic carcinogen and a promoting agent for thyroid carcinogenesis in rats. A total of 36 postpartum F344 rats were given drinking water containing AA at 0, 20, 40 or 80 ppm for 3 weeks during the lactation period, and their weaned offspring received the same AA-containing water for 3 more weeks. Offspring were then injected with N-methyl-N-nitrosourea (MNU; 40 mg/kg body weight, i.p.) once at week 7 after birth. Half the animals of the 0 and 40 ppm groups were additionally treated with the anti-thyroid agent sulfadimethoxine (SDM; 125 ppm) in the drinking water thereafter. Offspring were subjected to complete necropsy at week 50. All the major organs and macroscopic abnormalities were excised and examined histopathologically. There was no significant difference in the incidences of hyperplastic and neoplastic lesions in the target organs of AA and/or MNU, such as the brain, spinal cord, pituitary gland, thyroid, adrenal glands, uterus, mammary glands, clitoral gland and tunica vaginalis. In conclusion, no significant modifying actions of AA on MNU-induced multi-organ carcinogenesis were exhibited in any organs of rats when exposed prepubertally under the present experimental conditions.

We have reported that thyroid capsular thickening with inflammation induced by an antithyroidal agent, sulfadimethoxine (SDM), might play a role in the development of invasive follicular carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Inducible nitric oxide synthase (iNOS) expressed in the inflamed capsular regions further appeared to be implicated in the tumor progression. In the present study, the effects of an iNOS inhibitor, aminoguanidine (AG), on thyroid carcinogenesis were examined. F344 male rats were treated with SDM in drinking water (0.1%) with or without concomitant dietary administration of AG (0.2%) for 4 and 10 weeks after subcutaneous injection of DHPN at 2800 mg/kg bodyweight. At week 4, thyroid capsular thickening with inflammation was observed and iNOS-positive foci were found in the inflamed regions. In addition, single-strand DNA-positive inflammatory cells were scattered among neighboring follicular cells, indicating some cellular damage, at least partly in association with iNOS induction. Concurrent dietary administration of AG with SDM treatment slightly decreased the number of single-strand DNA-positive cells but did not alter the incidence and multiplicity of iNOS-positive foci in the inflamed capsular regions at week 4. At week 10, however, invasive follicular carcinomas predominantly arose in the thickened capsule in the DHPN-SDM-treated rats, and AG administration decreased (P < 0.05) their multiplicity. The carcinoma cells were partly positive for iNOS. These results thus suggested that iNOS induction in both inflammatory and tumor cells might play pivotal roles in tumor progression in this DHPN-SDM rat model.

NKX2-1 is a homeodomain transcription factor that is critical for genesis of the thyroid and transcription of the thyroid-specific genes. Nkx2-1-thyroid-conditional hypomorphic mice were previously developed in which Nkx2-1 gene expression is lost in 50% of the thyroid cells. Using this mouse line as compared with wild-type and Nkx2-1 heterozygous mice, a thyroid carcinogenesis study was carried out using the genotoxic carcinogen N-bis(2-hydroxypropyl)-nitrosamine (DHPN), followed by sulfadimethoxine (SDM) or the non-genotoxic carcinogen amitrole (3-amino-1,2,4-triazole). A significantly higher incidence of adenomas was obtained in Nkx2-1-thyroid-conditional hypomorphic mice as compared with the other two groups of mice only when they were treated with DHPN + SDM, but not amitrole. A bromodeoxyuridine incorporation study revealed that thyroids of the Nkx2-1-thyroid-conditional hypomorphic mice had >2-fold higher constitutive cell proliferation rate than the other two groups of mice, suggesting that this may be at least partially responsible for the increased incidence of adenoma in this mouse line after genotoxic carcinogen exposure. Thus, NKX2-1 may function to control the proliferation of thyroid follicular cells following damage by a genotoxic carcinogen.

Sulphonamides contamination of cultivated lands occurs through the recurrent spreading of animal wastes from intensive farming. The aim of this study was to test the effect(s) of sulphadimethoxine on the beneficial N-fixing Rhizobium etli-Phaseolus vulgaris symbiosis under laboratory conditions. The consequence of increasing concentrations of sulphadimethoxine on the growth ability of free-living R. etli bacteria, as well as on seed germination, seedling development and growth of common bean plants was examined. We have established that sulphadimethoxine inhibited the growth of both symbiotic partners in a dose-dependent manner. Bacterial invasion occurring in developing root nodules was visualized by fluorescence microscopy generating EGFP-marked R. etli bacteria. Our results proved that the development of symbiotic N-fixing root nodules is hampered by sulphadimethoxine thus identifying sulphonamides as toxic compounds for the Rhizobium-legume symbiosis: a low-input sustainable agricultural practice.

Spontaneously jaundiced Gunn rats exposed to sulfadimethoxine develop bilirubin encephalopathy (kernicterus) with hearing loss and dystonia, closely resembling the human syndrome. We recently characterized the electromyographic activity in this animal model supporting our clinical impression of dystonia. The objective of this study was to develop a simple, non-invasive method to quantify the motor deficits in dystonic rodents. On postnatal day 16, Gunn rats were treated with 100mg/kg of sulfadimethoxine or saline. On postnatal day 31, the ventral view of the animals was videotaped while the animals walked inside a Plexiglas chamber. Individual video frames were reviewed and specific gait parameters including hindlimb spread, step length ratio variability, stance/swing ratio and walking speed were compared between dystonic and non-dystonic jaundiced and non-jaundiced littermates. Data analysis demonstrated statistically significant increases in hindlimb spread and step length ratio variability and decreases in walking speed in dystonic animals as compared to controls. This study demonstrates a valuable technique to objectively characterize dystonia in Gunn rats, which could have wide use for other experimental movement disorders as well.

To identify differentially regulated molecules related to early and late stages of tumor promotion in a rat two-stage thyroid carcinogenesis model by an antithyroid agent, sulfadimethoxine, microarray-based microdissected lesion-specific gene expression profiling was carried out. Proliferative lesions for profiling were divided into two categories: (i) focal follicular cell hyperplasias (FFCH) and adenomas (Ad) as early lesions; and (ii) carcinomas (Ca) as more advanced. In both cases, gene expression was compared with that in surrounding non-tumor follicular cells. Characteristically, upregulation of cell cycle-related genes in FFCH + Ad, downregulation of genes related to tumor suppression and transcription inhibitors of inhibitor of DNA binding (Id) family proteins in Ca, and upregulation of genes related to cell proliferation and tumor progression in common in FFCH + Ad and Ca, were detected. The immunohistochemical distributions of molecules included in the altered expression profiles were further examined. In parallel with microarray data, increased localization of ceruloplasmin, cyclin B1, and cell division cycle 2 homolog A, and decreased localization of poliovirus receptor-related 3 and Id3 were observed in all types of lesion. Although inconsistent with the microarray data, thyroglobulin immunoreactivity appeared to reduce in Ca. The results thus suggest cell cycling facilitation by induction of M-phase-promoting factor consisting of cyclin B1 and cell division cycle 2 homolog A and generation of oxidative responses as evidenced by ceruloplasmin accumulation from an early stage, as well as suppression of cell adhesion involving poliovirus receptor-related 3 and inhibition of cellular differentiation regulated by Id3. Decrease of thyroglobulin in Ca may reflect dedifferentiation with progression.

After prophylactic treatment of 50 calves with 62mgkg(-1)day(-1) of sulfadimethoxine (SDM) for five days, the levels of the drug over time were followed in feces, bedding and stable manure, and then in the soil of a manured field and surrounding drainage courses. Analysis were done by HPLC after applying to the different matrices a quick and simple extraction procedure. The half-life of the drug in bedding was very short (24h). In stable manure the degradation rate of the drug slowed down and the calculated half-life was 64 days, with 390microgkg(-1) of SDM still detectable after three months maturation. However, in a five months matured stable manure obtained from other groups of calves subjected to the same prophylactic treatment, levels of SDM were <50muicrokg(-1) (LOD of the analytical method). After field fertilization with this manure, no traces of SDM were found in soil (LOD 10microgkg(-1)) or in the water (LOD 2microgl(-1)) from the surrounding drainage courses. Using the internationally recognised DAPHTOXKIT-Ftrade mark, a SDM toxicity test toward Daphnia magna was performed in the range 10-100mgl(-1). The test gave negative results both after 24 and 48h, confirming that microcrustaceans are less sensitive than other models to the toxicity of antibacterials. However, based on data from other authors, concerning algal toxicity and microbial inhibition, and on the analytical results from the current field study, the calculated worst-case EC50/PEC ratio for SDM both in freshwater and in soil was still >1000.

We previously demonstrated that thyroid capsular inflammation induced by continuous treatment with the antithyroidal agent sulfadimethoxine is associated with development of invasive follicular cell carcinomas in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). The inflammatory changes are characterized by large numbers of macrophages and lymphocytes as well as fibroblasts and we hypothesized that it might be enhanced by interplay between macrophages and T cells. To clarify this hypothesis, a comparative study was conducted between athymic nude (rnu/rnu) rats and euthymic (rnu/+) littermates initiated with DHPN (2800 mg/kg, s.c.) followed by sulfadimethoxine treatment in drinking water (0.1%) for 10 weeks. In rnu/+rats, marked capsular thickening with inflammation was induced along with invasive follicular cell carcinomas (2.8 +/- 1.3/rat). In rnu/rnu rats, limited fibrous capsular thickening was noted with or without minimal inflammatory change, and the multiplicity of invasive carcinomas was significantly lower (1.1 +/- 1.0/rat, P < 0.01). Inducible nitric oxide synthase expression in the inflamed lesions was detected in three of 10 rnu/+rats but in none of the rnu/rnu animals. The results thus suggest that development of invasive carcinomas is enhanced by capsular inflammation mediated by T cells, and inducible nitric oxide synthase induction may play a role in tumor progression.

Objective: High levels of bilirubin are neurotoxic and may result in deafness or auditory neuropathy/auditory dyssynchrony (AN/AD). The jaundiced (jj) Gunn rat animal model of kernicterus has electrophysiologic and neuroanatomic abnormalities of brainstem auditory nuclei with normal cochlear microphonic recordings. We examined morphologic changes in the cochlea, spiral ganglion, and auditory nerve and relate these findings to current understanding of AN/AD., Methods: At 15 days of age, jj and nonjaundiced (Nj) littermates were injected with sulfadimethoxine (sulfa) and killed 3 days later by transcardial perfusion. Sections were cut through decalcified temporal bones, cochlear nerves, and auditory brainstem and processed for light and electron microscopy and immunohistochemical localization of calbindin-D and parvalbumin., Results: Spiral ganglion neurons were severely degenerated with a paucity of myelinated axons in jj animals. Electron microscopy of the intramodilar auditory nerve revealed a lack of large caliber axons in jj-sulfa versus Nj-sulfa controls. Large diameter degenerating axons were characterized by an electron-dense atrophied axis cylinder resembling an axonopathy., Conclusions: Our findings of abnormal spiral ganglion cells and selective loss of large, myelinated auditory nerve fibers with no abnormalities in cochlear hair cells, support the sulfa-treated jj Gunn rat as a model for bilirubin induced AN/AD. The paucity of large caliber neurons undermines temporal coding of auditory information and neural synchrony and demonstrates that in addition to brainstem auditory nuclei, spiral ganglion neurons are selectively vulnerable to bilirubin toxicity.

The specificity of copromotion effects of caffeine with known goitrogenic factors on thyroid carcinogenesis was examined in rats pretreated with N-bis(2-hydroxypropyl)nitrosamine (DHPN). Male F344 rats were divided into 8 groups, each consisting of 10 animals, and received a single sc injection of 2,800 mg/kg DHPN. From one week after the DHPN initiation, they were given basal diet, iodine deficiency (ID) diet, 500 ppm phenobarbital (PB) solution or 1,000 ppm sulfadimethoxine (SDM) solution with or without 1,500 ppm caffeine feeding for 12 weeks. The caffeine, PB, SDM, and ID treatments significantly (p < 0.05 or 0.01) increased the relative thyroid weights, and the increases with PB or ID were further (p < 0.05 or 0.01) enhanced in combination with caffeine. SDM drastically promoted thyroid carcinogenesis in association with increased serum TSH levels regardless of the caffeine treatment. Thyroid follicular carcinomas and adenomas were more frequently observed in the additional caffeine groups than in the ID alone groups. The incidence and multiplicity of focal thyroid follicular hyperplasias in the ID-treated groups were significantly (p < 0.05 and 0.01) elevated in the case of combination with caffeine. Increases in serum TSH levels with PB or ID were also further enhanced in combination with caffeine. Serum thyroid hormone levels were significantly (p < 0.01) decreased by SDM but significantly (p < 0.05 or 0.01) increased by caffeine, PB or ID. Our results clearly indicate that dietary caffeine at a high dose of 1,500 ppm interacts with ID, but neither SDM nor PB, to promote rat thyroid carcinogenesis although the combined caffeine + PB treatment somewhat affected thyroid weights as well as thyroid hormone levels.

A 2-stage thyroid follicular carcinogenesis model in rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN) is widely used to detect modifying effects of chemicals on thyroid carcinogenesis. A number of goitrogens are known to strongly promote carcinogenesis, and the carcinomas often originate adjacent to the thyroid capsule and show invasive growth into the capsule or adjacent tissues. To clarify mechanisms of progression to invasive carcinomas, we sequentially evaluated histopathological and immunohistochemical characteristics of thyroids in male F344 rats treated with sulfadimethoxine (SDM, 0.1% in drinking water) for 0-10 weeks beginning 1 week after DHPN initiation (2800 mg/kg body weight, single s.c. injection). In DHPN-SDM-treated rats, multiple focal hyperplasias and adenomas developed in thyroid follicular parenchyma at weeks 4 to 6. Apart from the proliferative lesions, capsular thickening with inflammatory cell infiltration, mainly consisting of macrophages, and migration of follicular epithelium into the capsule were also observed. Focal hyperplasias/adenomas adjacent to the capsule progressively developed to invasive carcinomas at weeks 6 to 10. In thyroid parenchyma, malignant lesions were seldom observed. With SDM-treatment alone, although no neoplastic lesions were observed, capsular thickening with inflammation and epithelial migration resulted in intracapsular residual follicles. Intracapsular residual follicular cells as well as invasive and intrathyroidal carcinoma cells generally showed increased cell proliferative activity, coincidental with cytoplasmic/nuclear positivity for beta-catenin. These results suggested that beta-catenin activation related to capsular inflammation may play a role in development of invasive carcinomas but is insufficient for tumor formation by itself. Whether this is associated with mutations in the beta-catenin gene remains to be clarified.

Brainstem auditory evoked potentials (BAEPs) are a sensitive indicator of bilirubin neurotoxicity. Somatosensory evoked potentials (SEPs) have been proposed as another measure of toxicity, though the lemniscal pathways that generate the SEP are not involved in kernicterus. In 16 to 17-d-old jaundiced (jj) Gunn rats, serial BAEPs and SEPs were obtained up to 8 h after acute bilirubin toxicity. jjs were injected with 150 mg/kg sulfadimethoxine to displace bilirubin into brain tissue (n = 8); littermate controls were jjs given saline (n = 4) and nonjaundiced given sulfadimethoxine or saline (n = 7). After anesthesia, BAEP and SEP recordings were obtained at baseline and serially after injection. SEPs to median nerve stimulation were recorded from surface electrodes over the brachial plexus (Erb's) and contralateral parietal cortex, and subtracted to obtain central conduction time (CCT). There were no statistically significant different baseline values between groups. Baseline BAEP I, I-II, and I-III were 1.22 +/- 0.13, 1.11 +/- 0.12, and 2.10 +/- 0.15 ms, and SEP Erb's and CCT were 1.48 +/- 0.20 and 5.59 +/- 0.50 ms, respectively (n = 19). At 6.8 +/- 1.5 h after injection BAEP I, I-II, and I-III increased 0.10 +/- 0.08, 0.18 +/- 0.17, and 0.56 +/- 0.33 ms over baseline, respectively (p = 0.005, 0.01, and 0.001, respectively, paired, 1-tailed t-tests), in experimental but not control groups. SEP Erb's decreased slightly, -0.06 +/- 0.04 ms in experimental and -0.08 +/- 0.08 ms in control groups, while CCT did not change significantly. BAEPs were completely abolished in two jjs with no SEP changes. When injection of sulfonamide induced significant peripheral and central BAEP abnormalities in jaundiced rats, no SEP abnormalities occurred. SEPs assess proprioception but not other somatosensory function or sensory integration. The results demonstrate the selectivity of acute bilirubin toxicity for the auditory nervous system.

In order to improve the sensitivity of our previously established thyroid carcinogenesis model and to clarify whether endocrine disrupting chemicals with weak estrogenic activity have any modifying effects on the development of thyroid proliferative lesions, 6-week-old female castrated F344 rats were first given a single subcutaneous injection of 2000 mg/kg body weight of N-bis(2-hydroxypropyl)nitrosamine. From 1 week later, they received diets with: no supplement (basal diet (BD) group); cholesterol pellets containing 0.5 mg 17 beta-estradiol 3-benzoate (EB); or diet admixed with 1000 ppm methoxychlor (MXC) or 10,000 ppm bisphenol A (BPA) for 20 weeks. Furthermore, additional groups were administered 200 ppm sulfadimethoxine (SDM) in the drinking water simultaneously with the BD, EB, MXC or BPA treatments. Thyroid follicular cell hyperplasias, adenomas and/or carcinomas were induced only in the EB+SDM group, the incidences of non-malignant lesions being significantly increased, as compared with the BD+SDM group values. Furthermore, the serum level of thyroid stimulating hormone (TSH) was significantly increased in this group. No significant variation in quantitative values for thyroid proliferative lesions or TSH levels were observed in the other treated groups. The results of the present study convincingly indicate that EB, with strong estrogenic activity, but not MXC and BPA, with weak estrogenic activities, exerts promoting effects on thyroid carcinogenesis in rats. The present modified rat two-stage thyroid carcinogenesis model appears to have advantages over our previous model for screening purposes.

The specificity and dose dependence of the synergistic effects of soybean intake with iodine deficiency on the induction of thyroid proliferation were investigated in female F344 rats. In the first experiment, rats were divided into 6 groups, each consisting of 5 animals, and fed a basal diet containing 20% gluten, an iodine-deficient basal diet alone or an iodine-deficient diet containing 0.2%, 1.0%, 5.0% or 25% defatted soybean for 5 weeks. Soybean feeding synergistically induced thyroid hyperplasias with iodine deficiency only at the 25% dose. In the second experiment, rats were also divided into 6 groups, each consisting of 5 animals, and fed a basal diet, a diet containing 20% defatted soybean, 0.025% sulfadimethoxine (SDM), 20% defatted soybean + 0.025% SDM, 0.05% phenobarbital (PB) or 20% defatted soybean + 0.05% PB for 5 weeks. The SDM treatments significantly (P < 0.05 - 0.01) increased the thyroid weights, but this increase rate was less prominent in the SDM + soybean group than in the SDM alone group. The PB treatment was also associated with a tendency for increase in thyroid weight, but again this was smaller in the PB + soybean group than in the PB alone group. Although the SDM or PB treatments reduced the serum triiodothyronine and thyroxine levels and consequently increased the serum thyroid-stimulating hormone (TSH) levels, the soybean feeding did not affect or rather attenuated these changes. Our results clearly indicate that soybean feeding does not synergistically enhance the effects of SDM or PB on the rat thyroid. Thus it can be concluded that soybean intake specifically interacts with iodine deficiency in induction of thyroid proliferative lesions in rats, only at high doses.

The modifying effects of various environmental estrogenic compounds on thyroid carcinogenesis were investigated in a rodent two-stage carcinogenesis model. The compounds examined were a soy isoflavone mixture (SI) and genistein (GEN) as phytoestrogens, nonylphenol (NP) as a xenoestrogen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) as a thyroid carcinogen and sulfadimethoxine (SDM) as a known thyroid tumor promoter. Five-week-old male F344 rats were given a single subcutaneous injection of N-bis(2-hydroxypropyl)nitrosamine (DHPN; 2800 mg / kg, body weight) or the vehicle alone. Starting one week thereafter, GEN (250 or 25 ppm in diet), SI (400 ppm in diet), NP (250 or 25 ppm in diet), MX (30 ppm, in drinking water) or SDM (1000 ppm in drinking water) was administered for 12 weeks. Major organs including the thyroid, pituitary, liver, kidney, testis, brain and pancreas were weighed and histopathological observation was performed. Thyroid weights were significantly increased (P < 0.001) only in the SDM treatment groups, especially with DHPN pretreatment. Kidney weights were slightly increased in the NP or MX treatment groups, albeit without statistical significance. Histopathologically, thyroid proliferative lesions were only observed in the SDM alone or DHPN + SDM group with significant focal hyperplasias, adenomas and adenocarcinomas limited to the combined treatment case. There were no organ weight changes or histopathological lesions in the major organs including the thyroid in the GEN, SI, NP, and MX treatment groups regardless of DHPN pretreatment. Our results thus indicate that the weakly estrogenic compounds GEN, SI and NP and the environmental rat thyroid carcinogen MX do not exert any modifying effects on thyroid carcinogenesis in rats under the present experimental conditions.

Severe hyperbilirubinemia in neonates with prematurity and/or systemic illnesses such as hemolytic disease, acidosis, and hypoxemia enhances their risk for developing cerebral palsy, paralysis of ocular upgaze, and deafness. This neurologic syndrome has been associated with selective neuronal vulnerability in the basal ganglia, certain brainstem nuclei, and Purkinje cells. However, the mechanism by which bilirubin damages neurons remains unclear. In these studies, we found that intracerebral injection of N-methyl-D-aspartate (NMDA), an excitotoxic analogue of glutamate, caused greater injury in jaundiced 7-day-old Gunn (jj) rat pups than in nonjaundiced heterozygous (Nj) littermate controls. NMDA injection caused even greater injury when protein-bound bilirubin was displaced with the sulfonamide drug sulfadimethoxine in jaundiced homozygous pups. In additional experiments, the acute signs of bilirubin-mediated neuronal injury, induced in homozygous (jj) Gunn rats by treatment with sulfonamide, were reduced by concurrent treatment with the NMDA-type glutamate channel antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine (MK-801, dizocilpine). The results suggest that bilirubin may cause encephalopathy and neuronal injury, at least in part, through an NMDA receptor-mediated excitotoxic mechanism. This conclusion is consistent with clinical observations that bilirubin encephalopathy is synergistically worsened by hypoxemia, which also shares an excitotoxic mechanism of neuronal injury.

The purpose of this study was to investigate the process of micrognathic development in mouse fetus induced by Sulfadimethoxine (SDM). SDM of 3,000 mg/kg was administered orally to pregnant ICR mice on days 8, 9, 10, 11, 12, and 13 of gestation. The fetuses were removed on day 15 to analyze the incidence of micrognathia. The incidence of micrognathia was the highest in the treated group on day 10. The fetuses, SDM-treated on day 10, were sacrificed on days 11, 12, 13, 14, 15, and 18 and prepared for histological and morphological analyses. Histomorphological analyses were performed by lateral and horizontal graphic reconstruction using serial frontal sections. It was also performed by clearing and alcian blue-alizarin red double staining. In the treated group, bilateral sigmoid buckling and shortening of Meckel's cartilage became evident after day 14, however, the nasal septal and capsule cartilage were less affected. It was concluded that remarkable hypoplastic and malformed Meckel's cartilage, caused by the administration of SDM, induced mandibular dysmorphogenesis.

The homozygous (ii) Gunn rat provides a model for hyperbilirubinemia which includes prominent cerebellar hypoplasia. Development of the Gunn rat cerebellum was examined with and without the additional effects of elevating brain bilirubin concentration to still higher levels via sulfadimethoxine (sulfa) administration. Homozygous (jj) Gunn rats and heterozygous (Nj) littermate controls (n = 32 each) were given 100 mg/kg sulfa or saline at postnatal days 3, 7, 17, and 30, and most were sacrificed 24 h later (n = 4 for each genotype at each age). Cerebellar volume, total volume and cell number for each deep cerebellar nucleus, densities for Purkinje and granule cells in the cerebellar cortex of lobules II, VI and IX, and the density of vacuolated Purkinje cells were all measured quantitatively. Cytoplasmic vacuolation provided an indication of bilirubin toxicity and was never observed in the Nj control rats. Vacuolated Purkinje cells were first observed in jj-saline rats at 18 days and were found only in the more anterior lobules of the cerebellum (II and VI). By contrast, vacuolated Purkinje cells were observed in jj-sulfa rats at both 4 and 8 days, but only in the most posterior cerebellar lobule (IX). In all older jj rats, the decline in vacuolation was accompanied by significant necrosis and resorption of the Purkinje cells in the anterior lobules. Since the Purkinje cells in the posterior lobules are the first to differentiate in the cerebellum and are resistant to bilirubin toxicity in jj-saline rats, the results support the presence of a critical period when elevated brain bilirubin may be most toxic to neuronal development. The findings suggest that neurons undergoing differentiation at the time of bilirubin exposure are most susceptible to cell death, while cells that are slightly more or slightly less mature may show only transient changes.

In order to investigate whether goitrogens and liver enzyme-inducers modify the tumorigenesis in the liver or lung, 6-week old male F344 rats were given single subcutaneous injection of DHPN, and starting one week later received water containing goitrogens, namely sulfadimethoxine (SDM), propylthiouracil (PTU) and potassium thiocyanate (KSCN), or an enzyme-inducer, phenobarbital (PB), for 19 weeks ad libitum. Although the number of GST-P positive foci in the liver was significantly increased in the PB group as compared to the control group, there were no significant fluctuations in the SDM, PTU and PB groups. With respect to the lung, it is suggested that SDM, KSCN and PB may enhance the lung tumorigenesis, since the multiplicities of hyperplasias of alveolar epithelia were increased in groups treated with these compounds.

To determine whether production of thyroid proliferative lesions would be enhanced by intermittent rather than continuous treatment with a goitrogen, male F344 rats initiated with N-bis(2-hydroxypropyl)nitrosamine (DHPN, 2800 mg/kg body weight, single s.c. injection) were given water containing 0.1% sulfadimethoxine (SM) for 20 weeks (group 1) or 0.1% SM for the first 8 weeks followed by 2 cycles consisting of 2 weeks withdrawal and 4 weeks retreatment with 0.1% SM (group 2). Control rats (group 3) were untreated for 20 weeks after the DHPN initiation. Serum T3 and T4 levels were significantly decreased in groups 1 and 2 compared to group 3. Serum thyroid stimulating hormone level was significantly increased in all treated groups compared to group 3. The numbers of follicular cell hyperplasias were significantly increased in group 2 compared to group 1. BrdU labeling indices for follicular cells and hyperplasias were also significantly elevated in group 2 compared to group 1. Electron microscopic examination of thyrotrophs in the anterior pituitary in groups 1 and 2 revealed dilated rough ER cisternae with intracisternal dense granules. The number of intracytoplasmic secretory granules in group 2 was moderately decreased compared to group 1. Therefore, the results of the present study suggest that it may be possible to enhance production of thyroid neoplastic lesions by intermittent treatment.

Using a broth microdilution technique, the in vitro susceptibility of bacterial isolates from the equine respiratory tract to trimethoprim, sulfadoxine, sulfadimethoxine, and combinations of these compounds was determined. The bacterial strains (n = 88) isolated recently from horses with respiratory symptoms belonged to the following species: Streptococcus equi subsp. zooepidemicus (n = 34), Streptococcus equi subsp. equi (n = 22), Staphylococcus aureus (n = 9), Klebsiella pneumoniae (n = 7), Rhodococcus equi (n = 4), Pseudomonas spp. (n = 3) and Escherichia coli (n = 3). In addition, two isolates of Enterobacter spp. and one isolate of Streptococcus equisimilis, Staphylococcus intermedius, Proteus mirabilis and Serratia marcescens were examined. For determination of susceptibility of an organism the following minimal inhibitory concentrations (MIC) were fixed as limiting values: Trimethoprim < or = 0.5 microgram/ml, sulfadoxine < or = 32 micrograms/ml, sulfadimethoxine < or = 32 micrograms/ml, trimethoprim/sulfadoxine < or = 0.5/32 micrograms/ml, trimethoprim/sulfadimethoxine < or = 0.5/32 micrograms/ml. As expected, Rhodococcus-equi-isolates were resistant to the antimicrobials tested. However, most of the clinically more common isolates showed a high degree of susceptibility to the combinations. The fractional inhibitory concentration (FIC) indices indicated synergism of the combination-partners in a wide range. According to these in vitro results, application of trimethoprim/sulfonamide combinations for the initial therapy of equine respiratory tract infections can be recommended.

To examine changes in serum TSH and determine whether the sustained excess is necessary for the development and/or progression of thyroid tumors, male F344 rats were administered drinking water containing thiourea (TU), at 0.1 or 0.05%, or sulfadimethoxine (SM), at 0.025 or 0.0125%, for one week in Experiment I. All of the treated animals showed decreased serum levels of T3 and T4 and an increased TSH. In Experiment II, male rats were given a s.c. injection of N-bis(2-hydroxypropyl) nitrosamine (DHPN:1500 mg/kg BW) and, starting one week later, received drinking water containing the same doses of TU or SM as in Experiment I for the following 20 weeks. Thyroid follicular proliferative lesions were induced in most rats treated with TU and SM. However, these treated animals did not demonstrate any consistent alterations in serum T3, T4 and TSH levels, except for the high dose TU group. The present studies thus suggest that thyroid tumors can grow even under conditions of fluctuating serum TSH levels during the progression phase, although TSH stimulation might be an absolute requirement in the early phase of tumor development.

Neurologic and audiologic sequelae produced by bilirubin toxicity are preventable by appropriately timed therapeutic intervention. To understand the timing and reversibility of the neural dysfunction that follows exposure to bilirubin, we recorded brainstem auditory evoked potentials (BAEP) in the Gunn rat model of bilirubin encephalopathy. Abnormal BAEP occur in jaundiced Gunn rats after injection of sulfadimethoxine (sulfa) 100 mg/kg intraperitoneally, which displaces bilirubin from blood albumin binding sites and promotes the net transfer of bilirubin into brain tissue. Reversal of BAEP abnormalities with injection of human serum albumin (HSA) 2 g/kg intraperitoneally was studied in 17- to 20-d-old jaundiced Gunn rats. One animal from each of 14 litters was randomly assigned to one of the following treatment groups: 1) sulfa alone, 2) sulfa + HSA at 2 h, 3) sulfa + HSA at 8 h, or 4) saline alone. BAEP were recorded in each rat before and 0.1, 4, 8, 24, and 48 h after injection of sulfa or saline. BAEP I-II interwave intervals increased in all sulfa groups (p < 10(-9) to 0.27 ms (21%) above baseline at 8 h for the two sulfa groups not receiving treatment before that time (p = 0.0002), but increased less for the sulfa group given HSA at 2 h compared with untreated animals (p = 0.02). Partial recovery of function occurred at 24 and 48 h for both HSA-treated groups compared with their 8-h values (p = 0.001), and there was increased mortality at 24 h for the sulfa group not treated with HSA (p < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

No significant increases in serum SDH, ALT and AST activities were observed in goats and rats receiving oral sulfadimethoxine at 5 times the therapeutic dose. The quail showed significantly higher activities of SDH and ALT when compared to control values. Moderate increases in liver microsomal cytochrome P-450 and aniline hydroxylase activity were observed in goats and quail but no appreciable change in benzphetamine N-demethylase activity was detected in any species. These results suggest a lack of hepatic toxicity of sulfadimethoxine to these species under the reported experimental conditions.

Materials of an experimental study on allergenic properties of prolonged-action sulfanilamides and their semi-products are presented. Investigations involved the use of express-methods with intradermal administration of the substance into the ear and introduction of the agents together with the Freind adjuvant into the hind paw pad of a guinea pig. Experiments ascertained satisfactory dermal-allergic responses to the introduction of allergens differing in their strength by employing the above methods, as express ones for primary evaluation tof allergenic properties of drugs and their semi-products in conducting toxicological investigations.

Bilirubin toxicity produces significant neurologic and audiologic sequelae. Successful therapeutic intervention requires an understanding of the timing of neural dysfunction after exposure to bilirubin. BAEP were used in an animal model of bilirubin encephalopathy to study the onset of neural dysfunction after acute injection of a sulfonamide used to displace bilirubin out of the bloodstream and into tissue. Fourteen pairs of jaundiced Gunn rats from eight litters were studied at postnatal day 18. Baseline BAEP recordings were performed in anesthetized animals; then either sulfadimethoxine or an equal volume of saline was injected into the peritoneum. Another BAEP was done immediately, and then 2, 4, and 8 h after injection. Human serum albumin was injected into an additional 10 animals after the 2-h BAEP recording to see if induced BAEP abnormalities could be corrected. The sulfonamide-treated jj rats developed increased latencies for waves II and III, and I-II and I-III interwave intervals (p less than 0.0001). The latencies were prolonged by 2 h after injection and became progressively longer at 4 and 8 h. The amplitudes of waves II and III progressively decreased at 2, 4, and 8 h (p less than 0.0001). Latency and amplitude of waves I and IV did not change. The rats injected with albumin at 2 h showed improvement of BAEP abnormalities at 8 h. These studies show that neurophysiologic abnormalities occur as early as 2 h after intraperitoneal injection of sulfadimethoxine, and are reversible with appropriate therapy. These abnormalities are hypothesized to be due to the sulfonamide driven net transfer of free, toxic bilirubin into the central nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)