Combined toxicity of erythromycin and roxithromycin and their removal by Chlorella pyrenoidosa.

The ecological effects of antibiotics in surface water have attracted increasing research attention. In this study, we investigated the combined ecotoxicity of erythromycin (ERY) and roxithromycin (ROX) on the microalgae, Chlorella pyrenoidosa, and the removal of ERY and ROX during the exposure. The calculated 96-h median effect concentration (EC50) values of ERY, ROX, and their mixture (2:1 w/w) were 7.37, 3.54, and 7.91 mg∙L-1, respectively. However, the predicted EC50 values of ERY+ROX mixture were 5.42 and 1.51 mg∙L-1, based on the concentration addition and independent action models, respectively. This demonstrated the combined toxicity of ERY+ ROX mixture showed an antagonistic effect on Chlorella pyrenoidosa. During the 14-d culture, low-concentration (EC10) treatments with ERY, ROX, and their mixture caused the growth inhibition rate to decrease during the first 12 d and increase slightly at 14 d. In contrast, high-concentration (EC50) treatments significantly inhibited microalgae growth (p < 0.05). Changes in the total chlorophyll contents, SOD and CAT activities, and MDA contents of microalgae suggested that individual treatments with ERY and ROX induced higher oxidative stress than combined treatments. After the 14-d culture time, residual Ery in low and high concentration Ery treatments were 17.75% and 74.43%, and the residual Rox were 76.54% and 87.99%, but the residuals were 8.03% and 73.53% in ERY+ ROX combined treatment. These indicated that antibiotic removal efficiency was higher in combined treatments than that in individual treatments, especially at low concentrations (EC10). Correlation analysis suggested that there was a significant negative correlation between the antibiotic removal efficiency of C. pyrenoidosa and their SOD activity and MDA content, and the enhanced antibiotic removal ability of microalgae benefited from increased cell growth and chlorophyll content. Findings in this study contribute to predicting ecological risk of coexisting antibiotics in aquatic environment, and to improving biological treatment technology of antibiotics in wastewater.

Effects of erythromycin and roxithromycin on river periphyton: Structure, functions and metabolic pathways.

Macrolides have been frequently detected in the surface waters worldwide, posing a threat to the aquatic microbes. Several studies have evaluated the ecotoxicological effects of macrolides on single algal and bacterial strains. However, without considering the species interaction in the aquatic microbial community, these results cannot be extrapolated to the field. Thus, the present study aimed to evaluate the effects of two macrolides (erythromycin and roxithromycin) on the structure, photosynthetic process, carbon utilization capacity, and the antibiotic metabolic pathways in river periphyton. The colonized periphyton was exposed to the graded concentration (0 µg/L (control), 0.5 µg/L (low), 5 µg/L (medium), 50 µg/L (high)) of ERY and ROX, respectively, for 7 days. Herein, high levels of ERY and ROX altered the community composition by reducing the relative abundance of Chlorophyta in the eukaryotic community. Also, the Shannon and Simpson diversity indexes of prokaryotes were reduced, although similar effects were seldomly detected in the low and medium groups. In contrast to the unchanged carbon utilization capacity, the PSII reaction center involved in the periphytic photosynthesis was significantly inhibited by macrolides at high levels. In addition, both antibiotics had been degraded by periphyton, with the removal rate of 51.63-66.87% and 41.85-48.27% for ERY and ROX, respectively, wherein the side chain and ring cleavage were the main degradation pathways. Overall, this study provides an insight into the structural and functional toxicity and degradation processes of macrolides in river periphyton.

Aged microplastics change the toxicological mechanism of roxithromycin on Carassius auratus: Size-dependent interaction and potential long-term effects.

Size effects of microplastics have received extensive attention for their influence on other pollutants and harm to organisms. In this study, we investigated the uptake, elimination, tissue distribution and potential toxicity mechanism of roxithromycin (ROX) in the presence of 0.5, 5 and 50 µm of aged microplastics (AMPs) in Carassius auratus. The results showed that AMPs promoted the ROX bioaccumulation of various tissues in a size-dependent manner. AMPs and ROX significantly induced superoxide dismutase and catalase activities of liver and gut, and inhibited acetylcholinesterase activities of brain. The coexistence of smaller AMPs exacerbated pathological abnormalities in liver, gill and brain induced by ROX, while larger AMPs caused more intestinal damage. Moreover, high-throughput 16S rRNA gene sequencing indicated that the abundance of Proteobacteria in 0.5 µm AMPs and ROX joint treatments and Firmicutes and Bacteroidota in 50 µm AMPs and ROX joint treatments were significantly raised (p < 0.05). Metabolomics revealed that AMPs and ROX had a size-dependent long-term effect on gut microbial metabolites, which was mainly related to galactose metabolism, amino acid metabolism and primary bile acid biosynthesis pathways after a 7-day elimination, respectively. These results provide important insights into the relationship between the size effect of AMPs and interaction mechanism of AMPs and coexisting pollutants on aquatic organisms.

Intergenerational and biological effects of roxithromycin and polystyrene microplastics to Daphnia magna.

The influence of microplastics (MPs) on transgenerational effects of pharmaceuticals are drawing growing attention, however, whether aged process will alter the carrier effects of MPs were unknown. In this study, the intergenerational toxicity of single and combined exposure of polystyrene microplastics (PS-MPs) and roxithromycin (ROX) were investigated at the environmentally related concentrations, using Daphina magna as test organism. In the presence of UV-aged PS-MPs, the survival of D. magna for maternal generation (F0) at ROX concentration of 0.1 and 10 µg/L were increased by 20% and 40%, respectively. Meanwhile, the inhibition effects of ROX on the number of offspring and intrinsic rate of natural increase were obviously moderated. All these reproductive toxicity of ROX and PS-MPs in the first offspring (F1) were further aggravated both for the single and combined exposure. And the adverse effects disappeared much easier for the single exposure compared to the co-exposure through subsequent recovery. The combined exposure resulted in the change of inhibition of ROX on the swimming velocity and acceleration of D. magna into induction, while the feeding behavior kept inhibited. The AChE activity was distinctly increased by 1.61-3.25 times for the single and combined treatments, and the induction level of UV-aged MPs was higher than that of original MPs. Oxidative stress of the single exposure of ROX and original PS-MPs was observed with obvious induction of T-AOC and SOD activity, while the significant increase of MDA content was observed for the co-exposure. Among all indicators, the biochemical biomarkers and time of first brood were attributed to a class among all indicators, indicating that the time of first brood might be the most sensitive reproductive toxicity index. These results illustrated that both maternal impacts and offspring quality need to be considered for assessment of interaction of emerging contaminants.

Transcriptomic analysis suggests the inhibition of DNA damage repair in green alga Raphidocelis subcapitata exposed to roxithromycin.

Macrolide antibiotics are common contaminants in the aquatic environment. They are toxic to a wide range of primary producers, inhibiting the algal growth and further hindering the delivery of several ecosystem services. Yet the molecular mechanisms of macrolides in algae remain undetermined. The objectives of this study were therefore to: 1. evaluate whether macrolides at the environmentally relevant level inhibit the growth of algae; and 2. test the hypothesis that macrolides bind to ribosome and inhibit protein translocation in algae, as it does in bacteria. In this study, transcriptomic analysis was applied to elucidate the toxicological mechanism in a model green alga Raphidocelis subcapitata treated with 5 and 90 µg L-1 of a typical macrolide roxithromycin (ROX). While exposure to ROX at 5 µg L-1 for 7 days did not affect algal growth and the transciptome, ROX at 90 µg L-1 resulted in 45% growth inhibition and 2306 (983 up- and 1323 down-regulated) DEGs, which were primarily enriched in the metabolism of energy, lipid, vitamins, and DNA replication and repair pathways. Nevertheless, genes involved in pathways in relation to translation and protein translocation and processing were dysregulated. Surprisingly, we found that genes involved in the base excision repair process were mostly repressed, suggesting that ROX may be genotoxic and cause DNA damage in R. subcapitata. Taken together, ROX was unlikely to pose a threat to green algae in the environment and the mode of action of macrolides in bacteria may not be directly extrapolated to green algae.

Involvement of oxidative stress in the sensitivity of two algal species exposed to roxithromycin.

Algal species Raphidocelis subcapitata and Chlorella vulgaris are commonly used to test the chemicals with an antibacterial mode of action during marketing authorization process. However, significant differences in the sensitivity toward antibiotic exposure have been reported. The selection of an inappropriate test species would thus underestimate the environmental hazard of target chemicals and pose a potential threat to the ecosystem. Since oxidative stress is a crucial factor determining the inhibition of algal growth, an investigation on oxidative stress and antioxidant defense mechanisms in these two species was performed to explore its roles in species sensitivity. Here, roxithromycin (ROX), a macrolide antibiotic extensively used to treat respiratory, urinary and soft tissue infections, was used for testing. After 7 days exposure to ROX at the low (0.01 mg L-1) and high (0.09 mg L-1) concentrations, R. subcapitata was inhibited while the growth of C. vulgaris was stimulated. We investigated the roles of oxidative stress in algae by measuring the oxidative stress biomarkers (MDA), non-enzymatic antioxidants (GSH), and antioxidant enzymes (SOD, CAT, GP, GST). The results suggested that when the growth of algae is inhibited, MDA content as well as activities of oxidative stress enzymes would increase, and thus, activating the antioxidant system. On the contrary, it was inferred that when the growth is stimulated, MDA content and oxidative stress enzymes activities would decrease.

Interactive effects of roxithromycin and freshwater microalgae, Chlorella pyrenoidosa: Toxicity and removal mechanism.

Roxithromycin (ROX) has received increasing concern due to its large usage, ubiquitous detection in environment and high ecotoxicology risk. This study investigated the acute and chronic effects of ROX on the growth, chlorophyll, antioxidant enzymes, and malonaldehyde (MDA) content of Chlorella pyrenoidosa, as well as the removal mechanism of ROX during microalgae cultivation. The calculated 96 h median effective concentration of ROX on yield (EyC50) and specific growth rate (ErC50) of C. pyrenoidosa was 0.81 and 2.87 mg/L, respectively. After 96 h exposure, 1.0 ~ 2.0 mg/L of ROX significantly inhibited the synthesis of chlorophyll and promoted the activities of SOD and CAT (p < 0.05). The MDA content increased with the ROX concentration increasing from 0.5 ~ 1.0 mg/L, and then decreased to 105.76% of the control exposure to 2.0 mg/L ROX, demonstrating the oxidative damage could be moderated by the upregulation of SOD and CAT activities. During the 21 d chronic exposure, low concentration of ROX (0.1 and 0.25 mg/L) showed no significant effect on the growth and chlorophyll content of algae during the first 14 d, but significantly inhibited the growth of algae and the synthesis of chlorophyll at 21 d (p < 0.05 or p < 0.01). 1.0 mg/L ROX significantly inhibited the growth of microalgae during 3 ~ 21 d and the synthesis of chlorophyll at 7 ~ 21 d. High concentration and long-term exposure of low concentration of ROX caused the SOD and CAT activities and MDA content to increase, demonstrating a higher level of oxidative damage of microalgae. During the first 14 d, abiotic removal of ROX played a more important role, contributing about 12.21% ~ 21.37% of ROX removal. After 14 d, the biodegradation of ROX by C. pyrenoidosa gradually became a more important removal mechanism, contributing about 45.99% ~ 53.30% of ROX removal at 21 d. Bio-adsorption and bioaccumulation both played minor roles in the removal of ROX during algae cultivation.

Single and combined effects of microplastics and roxithromycin on Daphnia magna.

There is a rising concern about the pollution of microplastics (plastic particles < 5 mm) in water due to their physicochemical properties, especially their interaction with organic contaminants; however, such knowledge is still limited. The mass production and consumption of medication for the treatment of infectious diseases in human and animals have led to the ubiquity of antibiotics in the environment. We studied the single and joint effects of microplastics (1-µm and 10-µm polystyrene particles, PS) and roxithromycin (ROX) on Daphnia magna through the acute and sublethal toxicity tests. The 48-h median effective concentration (EC50) of 1-µm and 10-µm PS to D. magna was 66.97 mg/L and 199.94 mg/L, respectively, while the value of ROX was 20.28 mg/L. Malondialdehyde (MDA) levels and the activities of four enzymatic biomarkers, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione S-transferase (GST), were further detected to assess the oxidative stress caused in D. magna caused. The results showed that 48-h exposure to PS (0.1 mg/L) or ROX (0.01 mg/L) alone activated the activities of CAT and GST and MDA levels. When compared with the ROX alone, the responses of GPx and MDA in D. magna co-exposed to 1-µm PS were significantly decreased, while co-exposure to 10-µm PS significantly decreased the responses of GST and MDA. Furthermore, the integrated biomarker response version 2 (IBRv2) analysis revealed that co-exposure to 1-µm PS and ROX led to the strongest biological responses in D. magna. Our findings underlined that microplastics should be a concern when they interact with the co-existence of pollutants in the aquatic environment.

Degradation of typical macrolide antibiotic roxithromycin by hydroxyl radical: kinetics, products, and toxicity assessment.

The degradation of roxithromycin (ROX) by hydroxyl radical (·OH) generated by UV/H2O2 was systematically investigated in terms of degradation kinetics, effects of water chemistry parameters, oxidation products, as well as toxicity evaluation. The degradation of ROX by UV/H2O2 with varying light irradiation intensity, initial ROX concentration, and H2O2 concentration in pure water and wastewater all followed pseudo-first-order kinetics. The second-order rate constant for reaction between ROX and ·OH is 5.68 ± 0.34 × 109/M/s. The degradation rate of ROX increased with the pH; for instance, the apparent degradation rates were 0.0162 and 0.0309/min for pH 4 and pH 9, respectively. The presence of natural organic matter (NOM) at its concentrations up to 10 mg C/L did not significantly affect the removal of ROX. NO3- and NO2- anions inhibited the degradation of ROX due to the consumption of ·OH in reactions with these ions. Fe3+, Cu2+, and Mg2+ cations inhibited the degradation of ROX, probably because of the formation of ROX-metal chelates. A total of ten degradation products were tentatively identified by HPLC/LTQ-Orbitrap XL MS, which mainly derived from the attack on the oxygen linking the lactone ring and the cladinose moiety, tertiary amine and oxime side chain moiety by ·OH. The toxicity evaluation revealed that UV/H2O2 treatment of ROX induced the toxicity to bioluminescent bacteria increased.

New insight into the toxic effects of chloramphenicol and roxithromycin to algae using FTIR spectroscopy.

Antibiotics have been frequently detected in the aquatic environment, and they may affect aquatic organisms such as algae. Here we investigated toxicity of chloramphenicol (CAP) and roxithromycin (ROX) on four species of green algae (Pseudokirchneriella subcapitata, Scenedesmus quadricauda, Scenedesmus obliquus, and Scenedesmus acuminatus) at biochemical level by Fourier transform infrared spectroscopy (FTIR). The results revealed that both CAP and ROX had negative effects on algal growth and caused alterations of biochemical components. The toxic effects varied among the four algal species and S. acuminatus was found to be less sensitive than the other three species to the antibiotics. Even with similar mechanism of action, ROX displayed more adverse effects to algae than CAP. Both antibiotics could affect algae by inhibiting fatty acid synthesis and promoting protein and DNA aggregation, thus leading to accumulation of lipid peroxidation products, increment of the loose ß-sheet structure protein and transformation of B-DNA to Z-DNA. The findings from this study revealed the toxic mechanism of antibiotics to algae at the biochemical level.

Microbiome-related metabolite changes in gut tissue, cecum content and feces of rats treated with antibiotics.

The metabolic functionality of the gut microbiota contributes to the metabolism and well-being of its host, although detailed insight in the microbiota's metabolism is lacking. Omics technologies could facilitate unraveling metabolism by the gut microbiota. In this study, we performed metabolite profiling of different matrices of the gut, after antibiotic treatment of rats in order to evaluate metabolite changes observed at different dose levels and in different sexes, and to identify the best tissue matrix for further investigations regarding an assessment of metabolic effects of new compounds with antibiotic activity. Three different antibiotics (vancomycin, streptomycin and roxithromycin) were administered orally to rats for 28 days according to the OECD 407 guideline with a subsequent metabolic profiling in feces, cecum content and gut tissue (jejunum, ileum, cecum, colon and rectum). The data were analyzed in the MetaMap®Tox database. Treatment-related effects could be observed in the metabolite profile of feces and cecum content, but not of the different gut tissues. The metabolite profile showed compound specific effects on the microbiome. In line with the activity spectra of the antibiotics tested, vancomycin showed the largest effects, followed by roxithromycin and then by streptomycin for which changes were modest. In general, for all antibiotics the largest changes were observed for the classes of lipids (increase up to 94-fold), bile acids (increase up to 33-fold), amino acids (increase up to 200-fold) and amino acid related (increase up to 348-fold). The most relevant changes in metabolite values were similar in feces and cecum content and among sexes. The results of this targeted analysis indicate that the metabolic profiles of male and female animals in the gut microbiome are comparable. Concluding, taking other samples than feces does not add any extra information. Thus, as a non-invasive sampling method, feces provide a suitable matrix for studies on metabolism by the gut microbiota.

In vitro cytogenetic evaluation of the particular combination of flurbiprofen and roxithromycin.

Flurbiprofen (FLB) (anti-inflammatory and analgesic drug) and roxithromycin (RXM) (antibiotic) were widely used in world wide. This study deals with investigation of genotoxicity, cytotoxicity, and oxidative stress effects of a particular combination of these drugs in human cultured lymphocytes. Also, DNA damaging-protective effects of combination of these drugs were analyzed on plasmid DNA. Human lymphocytes were treated with different concentrations (FLB + RXM; 10 µg/mL + 25 µg/mL, 15 µg/mL + 50 µg/mL, and 20 µg/mL + 100 µg/mL) of the drugs following by study of their genotoxic and cytotoxic effects by analysis of cytokinesis-block micronucleus test and nuclear division index, respectively. The effect of the combination in aspect of anti-oxidative and DNA damaging activity was evaluated on Pet-22b plasmid. According to our results, the combination of FLB and RXM did not show a notable genotoxic effect on cells. Although each of the substances had been shown as a cytotoxic agent by previous researchers, in this research, the combination of these drugs did not exhibit any adverse effect on cell division. FLB had DNA protection effect against H2O2 while in combination with RXM had not the same effect on the plasmid.

Removal of pharmaceutically active compounds (PhACs) and toxicological response of Cyperus alternifolius exposed to PhACs in microcosm constructed wetlands.

This study investigated the effects of selected four pharmaceutically active compounds (PhACs) (carbamazepine, sulfamethoxazole, ofloxacin, and roxithromycin) on the photosynthesis and antioxidant enzymes of Cyperus alternifolius in constructed wetlands (CWs). Moreover, the removal and kinetics of PhACs in CWs were evaluated to explore the related removal mechanisms. Results showed that C. alternifolius can uptake and withstand certain PhACs. The PhAC tolerance of C. alternifolius might be attributed to their capacity to maintain relatively normal photosynthetic activity and elevated antioxidative defense. CWs offered comparable or even higher removal efficiencies for the selected PhACs compared with conventional WWTPs. The removal of the target PhACs was enhanced in the planted CWs versus the unplanted CWs mostly because of plant uptake and rhizosphere effects. In particular, carbamazepine, which is considered the most recalcitrant of the PhACs, was significantly reduced (p<0.05). The removal of target PhACs fitted into two distinct periods. The initial fast step (within the first 2 h) was essentially attributed to the adsorption onto the CW medium surface. The subsequent slow process (2-12 h) closely followed first-order kinetics probably because of the interaction between microorganisms and plants. The obtained results indicate that C. alternifolius can phytoremediate PhAC-contaminated waters in CWs.

Roxithromycin reduces the viability of cultured airway smooth muscle cells from a rat model of asthma.

OBJECTIVES: The purpose of this study was to investigate the effect of roxithromycin on apoptosis of airway smooth muscle cells (ASMCs) from a rat model of asthma and uncover signaling pathway underlying the cytotoxicity of roxithromycin. MATERIALS AND METHODS: ASMCs were isolated from a rat model of asthma and treated with or without roxithromycin for 48 h before parameter detection. Cell viability was assessed by WST-8 assay and flow cytometry after Annexin V/PI double staining. Changes in the mitochondrial membrane potential (ΔΨm) were measured by flow cytometry using JC-1. Cytochrome C (Cyt c), cleaved Caspase-9/3 and P27 were evaluated by Western Blot. RESULTS: Incubation with roxithromycin reduced ASMCs proliferation and enhanced apoptosis in a dose-dependent manner. Flow cytometry revealed a loss of ΔΨm and Western Blot displayed Caspase-9/3 activation as well as Cyt c release from mitochondria to the the cytosol after the treatment of roxithromycin. In addition, P27 were more strongly expressed in AMSCs treated with roxithromycin compared with the control group. CONCLUSIONS: Roxithromycin induced apoptosis of ASMCs derived from a rat model of asthma in a dose-dependent manner via a caspase-3- and caspase-9-dependent mitochondrial pathway, involving the up-regulation of P27.

Bioconcentration, metabolism, and biomarker responses in freshwater fish Carassius auratus exposed to roxithromycin.

To investigate the distribution, bioconcentration, metabolism, and biomarker responses of macrolide antibiotic roxithromycin (ROX) in fish, crucian carp (Carassius auratus) were exposed to various concentrations of ROX (4, 20, and 100µgL(-1)) for 20d. The ROX content in different tissues was quantified using UPLC/MS/MS. The liver exhibited the highest ROX concentration followed by the bile, gills, and muscle tissues. After 15d of exposure to different concentrations of ROX, the bioconcentration factors were 2.15-38.0 in the liver, 0.950-20.7 in the bile, 0.0506-19.7 in the gill, and 0.0439-13.8 in the muscle; these results were comparable to the estimated BCF values. The metabolites formed in the bile were identified based on metabolic identification in human bile. Additionally, the biomarkers, including acetylcholinesterase in the brain, as well as 7-ethoxyresorufin O-deethylase and superoxide dismutase in the liver changed significantly after 5, 10, 15, and 20d of exposure (P<0.05). Our results suggest that ROX can accumulate and be metabolized in fish; therefore, interactions between ROX or its metabolites and the biological systems may induce biochemical disturbances in fish.

Evidencing generation of persistent ozonation products of antibiotics roxithromycin and trimethoprim.

The mechanism of product formation during ozonation of two widely used antimicrobial agents, macrolide roxithromycin and inhibitor of dihydrofolate reductase (DHFR) trimethoprim was studied in laboratory-scale experiments with two types of matrix: distilled water and secondary wastewater effluent The structures ofthe primary and secondary reaction intermediates were elucidated byquadrupole-time-of-flight (QqToF) instrument, showing that in spite of their high ozone affinity both roxithromycin and trimethoprim oxidation pathway involve to a great degree the *OH radical chain reactions. In total nine ozonation products were detected, whereas two products of roxithromycin exhibited high refractoriness to ozonation, especially in the case of distilled water. Furthermore, the intact tertiary amine moiety of roxithromycin in these products suggests that the antimicrobial activity of the parent compound will be preserved.

Occurrences and ecological risks of roxithromycin, trimethoprim, and chloramphenicol in the Han River, Korea.

We evaluated the occurrence of three antibiotics (roxithromycin, trimethoprim, and chloramphenicol) in surface water and effluents from several sewage treatment plants (STPs) on the Han River, Korea. In addition, standard acute toxicity tests were conducted using the microbe Vibrio fischeri, freshwater macroinvertebrates Daphnia magna and Moina macrocopa, and fish (Oryzias latipes) for these antibiotics. Antibiotics were more frequently detected at higher amounts in effluent samples and in samples collected during the low-flow season. For trimethoprim and chloramphenicol, the levels observed in surface water during low flow were, on average, 108 and 31 ng/L, respectively. These levels were comparable to those measured in the municipal effluents (average, 80 and 37 ng/L, respectively), suggesting the presence of other sources upstream (e.g., livestock wastes). For roxithromycin, surface water levels were approximately an order of magnitude lower than effluent levels. Adverse effects of roxithromycin, trimethoprim, and chloramphenicol were observed at mg/L levels in standard acute aquatic ecotoxicity tests. Based on hazard quotients calculated for the three antibiotics, minimal risks to aquatic systems are suggested. To further increase scientific understanding about the potential impacts of these pharmaceuticals in the environment, however, chronic ecotoxicology studies, with more subtle but ecologically meaningful end points or in combination with other mechanistically related contaminants, may be beneficial.

Effects of eight antibacterial agents on cell survival and expression of epithelial-cell- or cell-adhesion-related genes in human gingival epithelial cells.

OBJECTIVE AND BACKGROUND: Our previous studies suggest that little adverse effect on the growth of the periodontal ligament would be expected, if tetracycline, minocycline, ofloxacin, roxithromycin, clarithromycin, and azithromycin were topically administered to the periodontal pocket at their MIC90 doses required to inhibit the growth of 90% of periodontopathic bacteria, including Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans. In the present study, we investigated the cytocidal effects of eight antibacterial agents on the human gingival epithelial cell line NDUSD-1. We also used NDUSD-1 cells to examine the effects of these agents on the mRNA and protein expressions of genes associated with the proliferation, differentiation, or cellular adhesion important to the epithelial regeneration of the periodontal attachment. METHODS: The cytocidal effect of the test agents was measured as a decrease in cell survival. To obtain a quantitative measure of the cytocidal effect, the LD50, i.e. the concentration which results in a 50% decrease in cell survival relative to the controls, was extrapolated from the concentration-response curves. The effects of the agents on the mRNA and protein expressions in NDUSD-1 cells were studied by reverse transcription-polymerase chain reaction (RT-PCR) and western blot analyses, respectively. RESULTS: The cytocidal effect increased in a concentration-dependent manner as the concentration of each of the eight test agents increased. The order of the agents according to their cytocidal effects (LD50) was minocycline > tetracycline > enoxacin > clarithromycin > roxythromycin approximately ofloxacin > azithromycin > erythromycin. The cytocidal effects of minocycline, tetracycline, enoxacin, clarithromycin, roxythromycin, ofloxacin, and azithromycin ranged from 1.2 to 23.2 times greater than that of erythromycin. The maximum non-cytocidal concentrations (MNCCs) of these agents for NDUSD-1 cells were: 0.3 microm for minocycline, 1 microm for tetracycline, 3 microm for ofloxacin and erythromycin, 10 microm for enoxacin, clarithromycin, and azithromycin, and 100 microm for roxythromycin. The MNCCs of ofloxacin, azithromycin, clarithromycin, and roxythromycin were greater than their MIC90 concentrations for periodontopathic bacteria described above. The effects on the mRNA and protein expressions of epithelial-cell- or cell-adhesion-related genes were examined in NDUSD-1 cells exposed to clarithromycin, roxythromycin, ofloxacin, and azithromycin at their MNCCs. None of the agents affected the mRNA expressions of five genes: keratinocyte growth factor receptor, keratin 18, integrin beta1, integrin beta4, and laminin 5gamma2. Clarithromycin and ofloxacin slightly decreased the protein expression of integrin beta4. Roxythromycin markedly decreased the protein expressions of integrin beta4 and laminin 5gamma2. Azithromycin had little inhibitory effects on the protein expressions of any of the five genes. CONCLUSIONS: These results suggest that little, if any, adverse effects on growth, differentiation, and adhesion of basal epithelial cells would be expected with topical administration of clarithromycin, ofloxacin or azithromycin to the periodontal pocket at a dose equivalent to the MIC90. It is important to note, however, that the extrapolation of these findings to in vivo conditions has yet to be undertaken.

Comparative pharmacodynamic analysis of Q-T interval prolongation induced by the macrolides clarithromycin, roxithromycin, and azithromycin in rats.

In order to evaluate the arrhythmogenic potency of macrolide antibiotics in a quantitative manner, we analyzed the influence of clarithromycin (CAM), roxithromycin (RXM), and azithromycin (AZM) on Q-T intervals from pharmacokinetic and pharmacodynamic points of view and in comparison with the potency of erythromycin (EM) previously reported by us for rats. Male Sprague-Dawley rats were anesthetized, and CAM (6.6, 21.6, and 43.2 mg/kg of body weight/h), RXM (20 and 40 mg/kg/h), and AZM (40 and 100 mg/kg/h) were intravenously injected for 90 min to obtain the time courses of drug concentrations in plasma and the changes in the Q-T intervals during and after the drug injections. Distinct Q-T interval prolongation of up to 10 ms was observed with CAM at its clinical concentrations. RXM and AZM evoked Q-T interval prolongation at concentrations higher than their clinical ranges. The potencies for Q-T interval prolongation, assessed as the slope of the concentration-response relationship, were 6.09, 0.536, and 0.989 ms. ml/microg for CAM, RXM, and AZM, respectively. There was hysteresis between the change in the Q-T intervals and the time course of the plasma concentration of each drug. The rank order of clinical arrhythmogenicity was estimated to be EM > CAM > RXM > AZM, as assessed from the present results and our previous report for EM. In conclusion, RXM and AZM were estimated to be less potent at provoking arrhythmia than EM and CAM. These results should be useful for making a safer choice of an appropriate agent for patients with electrocardiographic risk factors.

Cytotoxicity of macrolide antibiotics in a cultured human liver cell line.

Cytotoxicity of erythromycin base, erythromycin estolate, erythromycin-11,12-cyclic carbonate, roxithromycin, clarithromycin and azithromycin was compared in cultured human non-malignant Chang liver cells using reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and cellular protein concentration as end points of toxicity. Erythromycin estolate was the most toxic macrolide in all tests differing clearly from all the other macrolides studied. Erythromycin-11,12-cyclic carbonate was also more toxic than the other macrolides. Roxithromycin and clarithromycin were the next toxic derivatives, while erythromycin base and azithromycin were least toxic. Thus, cytotoxicity of the new semisynthetic macrolides, roxithromycin, clarithromycin and azithromycin, is not substantially different from that of erythromycin base. In view of the low level of hepatotoxicity of macrolides hitherto reported in humans, the results do not suggest any substantial risk for hepatic disorders related to the use of azithromycin, clarithromycin and roxithromycin.