Activation of RARα induces autophagy in SKBR3 breast cancer cells and depletion of key autophagy genes enhances ATRA toxicity

All-trans retinoic acid (ATRA), a pan-retinoic acid receptor (RAR) agonist, is, along with other retinoids, a promising therapeutic agent for the treatment of a variety of solid tumors. On the one hand, preclinical studies have shown promising anticancer effects of ATRA in breast cancer; on the other hand, resistances occurred. Autophagy is a cellular recycling process that allows the degradation of bulk cellular contents. Tumor cells may take advantage of autophagy to cope with stress caused by anticancer drugs. We therefore wondered if autophagy is activated by ATRA in mammary tumor cells and if modulation of autophagy might be a potential novel treatment strategy. Indeed, ATRA induces autophagic flux in ATRA-sensitive but not in ATRA-resistant human breast cancer cells. Moreover, using different RAR agonists as well as RARα-knockdown breast cancer cells, we demonstrate that autophagy is dependent on RARα activation. Interestingly, inhibition of autophagy in breast cancer cells by either genetic or pharmacological approaches resulted in significantly increased apoptosis under ATRA treatment and attenuated epithelial differentiation. In summary, our findings demonstrate that ATRA-induced autophagy is mediated by RARα in breast cancer cells. Furthermore, inhibition of autophagy results in enhanced apoptosis. This points to a potential novel treatment strategy for a selected group of breast cancer patients where ATRA and autophagy inhibitors are applied simultaneously.

Clinical relevance of DPYD variants c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity: a systematic review and meta-analysis of individual patient data

BACKGROUND The best-known cause of intolerance to fluoropyrimidines is dihydropyrimidine dehydrogenase (DPD) deficiency, which can result from deleterious polymorphisms in the gene encoding DPD (DPYD), including DPYD*2A and c.2846A>T. Three other variants-DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A-have been associated with DPD deficiency, but no definitive evidence for the clinical validity of these variants is available. The primary objective of this systematic review and meta-analysis was to assess the clinical validity of c.1679T>G, c.1236G>A/HapB3, and c.1601G>A as predictors of severe fluoropyrimidine-associated toxicity. METHODS We did a systematic review of the literature published before Dec 17, 2014, to identify cohort studies investigating associations between DPYD c.1679T>G, c.1236G>A/HapB3, and c.1601G>A and severe (grade ≥3) fluoropyrimidine-associated toxicity in patients treated with fluoropyrimidines (fluorouracil, capecitabine, or tegafur-uracil as single agents, in combination with other anticancer drugs, or with radiotherapy). Individual patient data were retrieved and analysed in a multivariable analysis to obtain an adjusted relative risk (RR). Effect estimates were pooled by use of a random-effects meta-analysis. The threshold for significance was set at a p value of less than 0·0167 (Bonferroni correction). FINDINGS 7365 patients from eight studies were included in the meta-analysis. DPYD c.1679T>G was significantly associated with fluoropyrimidine-associated toxicity (adjusted RR 4·40, 95% CI 2·08-9·30, p<0·0001), as was c.1236G>A/HapB3 (1·59, 1·29-1·97, p<0·0001). The association between c.1601G>A and fluoropyrimidine-associated toxicity was not significant (adjusted RR 1·52, 95% CI 0·86-2·70, p=0·15). Analysis of individual types of toxicity showed consistent associations of c.1679T>G and c.1236G>A/HapB3 with gastrointestinal toxicity (adjusted RR 5·72, 95% CI 1·40-23·33, p=0·015; and 2·04, 1·49-2·78, p<0·0001, respectively) and haematological toxicity (adjusted RR 9·76, 95% CI 3·03-31·48, p=0·00014; and 2·07, 1·17-3·68, p=0·013, respectively), but not with hand-foot syndrome. DPYD*2A and c.2846A>T were also significantly associated with severe fluoropyrimidine-associated toxicity (adjusted RR 2·85, 95% CI 1·75-4·62, p<0·0001; and 3·02, 2·22-4·10, p<0·0001, respectively). INTERPRETATION DPYD variants c.1679T>G and c.1236G>A/HapB3 are clinically relevant predictors of fluoropyrimidine-associated toxicity. Upfront screening for these variants, in addition to the established variants DPYD*2A and c.2846A>T, is recommended to improve the safety of patients with cancer treated with fluoropyrimidines. FUNDING None.

Acute Toxicity and Quality of Life After Dose-Intensified Salvage Radiation Therapy for Biochemically Recurrent Prostate Cancer After Prostatectomy: First Results of the Randomized Trial SAKK 09/10

PURPOSE Patients with biochemical failure (BF) after radical prostatectomy may benefit from dose-intensified salvage radiation therapy (SRT) of the prostate bed. We performed a randomized phase III trial assessing dose intensification. PATIENTS AND METHODS Patients with BF but without evidence of macroscopic disease were randomly assigned to either 64 or 70 Gy. Three-dimensional conformal radiation therapy or intensity-modulated radiation therapy/rotational techniques were used. The primary end point was freedom from BF. Secondary end points were acute toxicity according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0) and quality of life (QoL) according to the European Organisation for Research and Treatment of Cancer Quality of Life Questionnaires C30 and PR25. RESULTS Three hundred fifty patients were enrolled between February 2011 and April 2014. Three patients withdrew informed consent, and three patients were not eligible, resulting in 344 patients age 48 to 75 years in the safety population. Thirty patients (8.7%) had grade 2 and two patients (0.6%) had grade 3 genitourinary (GU) baseline symptoms. Acute grade 2 and 3 GU toxicity was observed in 22 patients (13.0%) and one patient (0.6%), respectively, with 64 Gy and in 29 patients (16.6%) and three patients (1.7%), respectively, with 70 Gy (P = .2). Baseline grade 2 GI toxicity was observed in one patient (0.6%). Acute grade 2 and 3 GI toxicity was observed in 27 patients (16.0%) and one patient (0.6%), respectively, with 64 Gy, and in 27 patients (15.4%) and four patients (2.3%), respectively, with 70 Gy (P = .8). Changes in early QoL were minor. Patients receiving 70 Gy reported a more pronounced and clinically relevant worsening in urinary symptoms (mean difference in change score between arms, 3.6; P = .02). CONCLUSION Dose-intensified SRT was associated with low rates of acute grade 2 and 3 GU and GI toxicity. The impact of dose-intensified SRT on QoL was minor, except for a significantly greater worsening in urinary symptoms.

Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

The impact of ABCC11 polymorphisms on the risk of early-onset fluoropyrimidine toxicity

A missense variant (c.1637C>T, T546M) in ABCC11 encoding the MRP8 (multidrug resistance protein 8), a transporter of 5-fluorodeoxyuridine monophosphate, has been associated with an increased risk of 5-fluorouracil-related severe leukopenia. To validate this association, we investigated the impact of the ABCC11 variants c.1637C>T, c.538G>A and c.395+1087C>T on the risk of early-onset fluoropyrimidine-related toxicity in 514 cancer patients. The ABCC11 variant c.1637C>T was strongly associated with severe leukopenia in patients carrying risk variants in DPYD, encoding the key fluoropyrimidine-metabolizing enzyme dihydropyrimidine dehydrogenase (odds ratio (OR): 71.0; 95% confidence interval (CI): 2.5-2004.8; Pc.1637C>T*DPYD=0.013). In contrast, in patients without DPYD risk variants, no association with leukopenia (OR: 0.95; 95% CI: 0.34-2.6) or overall fluoropyrimidine-related toxicity (OR: 1.02; 95% CI: 0.5-2.1) was observed. Our study thus suggests that c.1637C>T affects fluoropyrimidine toxicity to leukocytes particularly in patients with high drug exposure, for example, because of reduced fluoropyrimidine catabolism.

STUDIES OF DISULFOTON TOLERANCE IN RATS

Disulfoton (O,O, diethyl S-2-(ethylthio)ethyl phosphorodithioate) and other organophosphorus ester compounds are insecticides which inhibit acetylcholinesterase. Chemicals of this class cause signs of toxicity in mammals which are referable to acculmulation of acetylcholine at neuroeffector sites. A tolerance to this toxic action can be induced in experimental animals by giving multiple, sublethal doses of the compounds. There is strong evidence that disulfoton tolerance occurs because of a reduction in the sensitivity of tissues in the affected animals to acetylcholine.^ Experiments were designed to test the possibility that a decrease in the number of muscarinic cholinergic receptors could be downmodulating the sensitivity of tissues to acetylcholine. It was found that, concomitant with the onset of disulfoton tolerance, there was a decrease relative to control values in the specific binding of {('3)H} quinuclidinyl benzilate ({('3)H}QNB, a compound which selectively labels muscarinic cholinergic receptors) to homogenates of rat brain and ileal muscle. The decrease in {('3)H}QNB binding was due to a reduction in the density of muscarinic receptors. There was, however, no alteration in the binding of {('3)H} QNB, or the muscarinic agonists {('3)H} oxotremorine-M and oxotremorine to atria from disulfoton-tolerant rats. The possibility that cardiac tissue was not subsensitive to cholinergic agonists was ruled out in experiments testing the effect of the muscarinic agonist carbachol on heart rate in vivo, and the negative chronotropic effect of oxotremorine on atria from disulfoton-tolerant rats: a clear reduction in the sensitivity to cholinergic agonists was seen in each case. It was, therefore concluded that the specificity and temporal correlation of {('3)H}QNB binding decreases suggested that the loss of muscarinic receptors might play a role in modulating the sensitivity of several tissues to acetylcholine, but that other mechanisms also contribute to the tolerance phenomenon.^ Other experiments revealed that disulfoton tolerance, as measured by resistance to the lethal effects of carbachol, could be induced by feeding rats low levels of the organophosphorus ester in the diet. The concentration of disulfoton used inhibited acetylcholinesterase, but not to the extent that overt signs of toxicity were observed. These results suggested that tolerance to organophosphorus ester insecticides could be induced in rodents with a dosing scheme which more closely modeled the sort of low level exposures which would be expected in humans environmentally or occupationally in contact with these compounds. ^

SYSTEMIC TOXICITY IN RODENTS FOLLOWING ACUTE AND SUBCHRONIC INHALATION OF FORMALDEHYDE (FLOW CYTOMETRY, CELL CYCLE KINETICS, ALKALINE ELUTION)

Systemic toxicity was evaluated in Sprague-Dawley (SD) rats and A-strain mice exposed to HCHO inhalation at 0, 0.5, 3, or 15 ppm for six hours/day, five days/week for up to 24 weeks. Toxicity was measured by flow cytometry to detect changes in cell cycle RNA and DNA content and by alkaline elution to detect DNA protein cross-link (DPC) formation.^ A G(,2)M block was detected in SD rat marrow following one week of exposure to 0.5, 3, or 15 ppm HCHO, but this block did not persist. No effect was noticed in mouse marrow. Only a minimal increase in RNA content was detected in rat or mouse marrow while exfoliated lung cells showed a significant increase in RNA activity after one week of exposure.^ Acute exposure in SD rats for four hours/day for one or three days at 150 ppm showed an increase in RNA activity in exfoliated lung cells but not in the marrow after one day. On the third day, dead cells were detected in exfoliated lung cells.^ In alkaline elution studies, no DPC were detected in marrow of SD rats after 24 weeks exposure up to 15 ppm. During acute exposures, a dose response relationship was detected in SD rat exfoliated lung cells which yielded cross-linking factors of 0.954, 1.237, and 1.417 following a four hour exposure to 15, 50, or 150 ppm, respectively. No DPC were detected in the marrow at 150 ppm. In vitro exposures to HCHO of CHO and SHE cells and rat marrow cells revealed the production of DPC and DNA-DNA cross-links.^ Cytoxan treatment of SD rats was used to provide positive controls for flow cytometry and alkaline elution. A drastic reduction in RNA content and cycling cells occurred one day following treatment. After four days, RNA content was greatly increased; and on day eleven the marrow had regenerated. DPCs were detected in both the marrow and the exfoliated lung cells.^ The lack of significant responses in SD rats and A-strain mice below 15 ppm HCHO is explainable by host defense mechanisms. Apparently, the mucociliary apparatus and enzymatic detoxification are sufficient to reduce systemic toxicity to low level concentrations of formaldehyde. ^

BENZENE TOXICITY AND THE OCCURRENCE OF BENZENE IN THE AMBIENT AIR OF THE HOUSTON AREA

This study was conducted by either literature review or actual field survey. Results are summarized as follows: (1) Long-term occupational exposure of workers to benzene vapor at levels of 3-7 ppm, 2-3 ppm and 1.6 ppm may result in a decreased level of leucocyte alkaline phosphates, an increased incidence of chromosome aberrations and an increased level of ALA in erythrocytes, respectively; (2) Benzene is capable of causing fetotoxic effects in animals at levels as low as 10 ppm by volume; (3) Exposure of animals to or less than 1 ppm benzene vapor may result in leucopenia, an inverse ratio of muscle antagonist chronaxy and a decreased level of ascorbic acid in fetus's and mother's liver as well as whole embryo; (4) Benzene is causally associated with the increased incidence of pancytopenia, including unicytopenia, bicytopenia and aplastic anemia, and chromosome aberrations in occupational exposure population, and at best benzene must also be considered as a leukemogen; (5) Since it can be emitted into the atmosphere from both man-made and natural sources, benzene in some concentrations is present everywhere in the various compartments of the environment; (6) The findings of the emission of benzene from certain natural sources indicate that reducing benzene to a zero-level of exposure is theoretically impossible; (7) The annual average of benzene concentration detected in the Houston ambient air is 2.50 ppb, which is about 2.4 times higher than the nation-wide annual average exposure level and may have been some health implications to the general public; (8) In the Houston area, stationary sources are more important than mobile sources in contributing to benzene in the ambient air. ^

Estudio preliminar de los efectos letales y sub-letales de extractos etanólicos de cuatro especies xerófitas del altiplano chileno contra Tetranychus cinnabarinus (Acarina: Tetranychidae)

Se evaluaron los efectos letales y subletales de extractos etanólicos de cuatro plantas originarias de la zona alto andina del desierto chileno (Baccharis tola Phil., Fabiana densa J. Remy, Lampayo medicinalis F. Phil. y Azorella compacta Phil.) sobre adultos y huevos de Tetranychus cinnabarinus (Boisduval) (Acari: Tetranychidae), en condiciones de laboratorio (26 ± 2°C, 70 ± 10% HR y fotoperíodo de 16:8 luz: oscuridad). Los mayores niveles de mortalidad por contacto directo sobre adultos fueron obtenidos por A. compacta al 10% (p/v), L. medicinalis 10% (p/v) y A. compacta 7,5% (p/v), los cuales no presentaron diferencias significativas (p<0,05) con respecto al tratamiento control (spirodiclofen). Las CL50 más bajas fueron obtenida por F. densa con 0,71% (p/v) y L. medicinalis con 0,98% (p/v). A. compacta presentó la CL90 más baja con 5,10% (p/v) y la mayor eficacia por obtener un coeficiente de 3,21 (CL90/CL50). Lampayo medicinalis al 10% (p/v) presentó el mayor porcentaje de huevos no eclosionados con un 73,15%. Baccharis tola al 7% (p/v) expuso la mayor toxicidad por efecto residual con una mortalidad de 57,8% sobre adultos de T. cinnabarinus. Fabiana densa mantuvo efecto repelente hasta las 72 horas. Lampayo medicinalis, B. tola y A. compacta perdieron levemente su actividad repelente en el tiempo.

Seawater carbonate chemistry and toxicity of Pseudo-nitzschia fraudulenta in a laboratory experiment

Anthropogenic CO2 is progressively acidifying the ocean, but the responses of harmful algal bloom species that produce toxins that can bioaccumulate remain virtually unknown. The neurotoxin domoic acid is produced by the globally-distributed diatom genus Pseudo-nitzschia. This toxin is responsible for amnesic shellfish poisoning, which can result in illness or death in humans and regularly causes mass mortalities of marine mammals and birds. Domoic acid production by Pseudo-nitzschia cells is known to be regulated by nutrient availability, but potential interactions with increasing seawater CO2 concentrations are poorly understood. Here we present experiments measuring domoic acid production by acclimatized cultures of Pseudo-nitzschia fraudulenta that demonstrate a strong synergism between projected future CO2 levels (765 ppm) and silicate-limited growth, which greatly increases cellular toxicity relative to growth under modern atmospheric (360 ppm) or pre-industrial (200 ppm) CO2 conditions. Cellular Si:C ratios decrease with increasing CO2, in a trend opposite to that seen for domoic acid production. The coastal California upwelling system where this species was isolated currently exhibits rapidly increasing levels of anthropogenic acidification, as well as widespread episodic silicate limitation of diatom growth. Our results suggest that the current ecosystem and human health impacts of toxic Pseudo-nitzschia blooms could be greatly exacerbated by future ocean acidification and 'carbon fertilization' of the coastal ocean.

Studying the effect of CO2-induced acidification on sediment toxicity using acute amphipod toxicity test

Carbon capture and storage is increasingly being considered one of the most efficient approaches to mitigate the increase of CO2 in the atmosphere associated with anthropogenic emissions. However, the environmental effects of potential CO2 leaks remain largely unknown. The amphipod Ampelisca brevicornis was exposed to environmental sediments collected in different areas of the Gulf of Cádiz and subjected to several pH treatments to study the effects of CO2-induced acidification on sediment toxicity. After 10 days of exposure, the results obtained indicated that high lethal effects were associated with the lowest pH treatments, except for the Ría of Huelva sediment test. The mobility of metals from sediment to the overlying seawater was correlated to a pH decrease. The data obtained revealed that CO2-related acidification would lead to lethal effects on amphipods as well as the mobility of metals, which could increase sediment toxicity.

The effects of elevated CO2 on the growth and toxicity of field populations and cultures of the saxitoxin-producing dinoflagellate, Alexandrium fundyense

The effects of coastal acidification on the growth and toxicity of the saxitoxin-producing dinoflagellate Alexandrium fundyense were examined in culture and ecosystem studies. In culture experiments, Alexandrium strains isolated from Northport Bay, New York, and the Bay of Fundy, Canada, grew significantly faster (16-190%; p < 0.05) when exposed to elevated levels of PCO2 ( 90-190 Pa=900-1900 µatm) compared to lower levels ( 40 Pa=400 µatm). Exposure to higher levels of PCO2 also resulted in significant increases (71-81%) in total cellular toxicity (fg saxitoxin equivalents/cell) in the Northport Bay strain, while no changes in toxicity were detected in the Bay of Fundy strain. The positive relationship between PCO2 enrichment and elevated growth was reproducible in natural populations from New York waters. Alexandrium densities were significantly and consistently enhanced when natural populations were incubated at 150 Pa PCO2 compared to 39 Pa. During natural Alexandrium blooms in Northport Bay, PCO2 concentrations increased over the course of a bloom to more than 170 Pa and were highest in regions with the greatest Alexandrium abundances, suggesting Alexandrium may further exacerbate acidification and/or be especially adapted to these acidi-fied conditions. The co-occurrence of Alexandrium blooms and elevated PCO2 represents a previously unrecognized, compounding environmental threat to coastal ecosystems. The ability of elevated PCO2 to enhance the growth and toxicity of Alexandrium indicates that acidification promoted by eutrophication or climate change can intensify these, and perhaps other, harmful algal blooms.

Effect of simulated ocean acidification on the acute toxicity of Cu and Cd to Tigriopus japonicus

Heavy metals pollution in marine environments has caused great damage to marine biological and ecological systems. Heavy metals accumulate in marine creatures, after which they are delivered to higher trophic levels of marine organisms through the marine food chain, which causes serious harm to marine biological systems and human health. Additionally, excess carbon dioxide in the atmosphere has caused ocean acidification. Indeed, about one third of the CO2 released into the atmosphere by anthropogenic activities since the beginning of the industrial revolution has been absorbed by the world's oceans, which play a key role in moderating climate change. Modeling has shown that, if current trends in CO2 emissions continue, the average pH of the ocean will reach 7.8 by the end of this century, corresponding to 0.5 units below the pre-industrial level, or a three-fold increase in H+ concentration. The ocean pH has not been at this level for several millions of years. Additionally, these changes are occurring at speeds 100 times greater than ever previously observed. As a result, several marine species, communities and ecosystems might not have time to acclimate or adapt to these fast changes in ocean chemistry. In addition, decreasing ocean pH has the potential to seriously affect the growth, development and reproduction reproductive processes of marine organisms, as well as threaten normal development of the marine ecosystem. Copepods are an important part of the meiofauna that play an important role in the marine ecosystem. Pollution of the marine environment can influence their growth and development, as well as the ecological processes they are involved in. Accordingly, there is important scientific value to investigation of the response of copepods to ocean acidification and heavy metals pollution. In the present study, we evaluated the effects of simulated future ocean acidification and the toxicological interaction between ocean acidity and heavy metals of Cu and Cd on T. japonicus. To accomplish this, harpacticoids were exposed to Cu and Cd concentration gradient seawater that had been equilibrated with CO2 and air to reach pH 8.0, 7.7, 7.3 and 6.5 for 96 h. Survival was not significantly suppressed under single sea water acidification, and the final survival rates were greater than 93% in both the experimental groups and the controls. The toxicity of Cu to T. japonicus was significantly affected by sea water acidification, with the 96h LC50 decreasing by nearly threefold from 1.98 to 0.64 mg/L with decreasing pH. The 96 h LC50 of Cd decreased with decreasing pH, but there was no significant difference in mortality among pH treatments. The results of the present study demonstrated that the predicted future ocean acidification has the potential to negatively affect survival of T. japonicus by exacerbating the toxicity of Cu. The calculated safe concentrations of Cu were 11.9 (pH 7.7) and 10.5 (pH 7.3) µg/L, which were below the class I value and very close to the class II level of the China National Quality Standard for Sea Water. Overall, these results indicate that the Chinese coastal sea will face a

Effects of sublethal doses of methoxyfenozide on the development, survival and reproduction of Spodoptera frugiperda.

The lethal and sublethal effects of the ecdysone agonist methoxyfenozide on the fall armyworm, Spodoptera frugiperda (J. E. Smith), were investigated by feeding a methoxyfenozide-treated diet to ϐifth instars until pupation in doses corresponding to the LC 10 and LC 25 for the compound. Larval mortality reached 8% and 26% in the low and high concentration groups, respectively, on the seventh day of the experiment. A progressive larval mortality of 12% for the LC 10 and 60% for the LC 25 was observed before pupation. Treated larvae exhibited lower pupal weights, higher pupal mortality, presence of deformed pupae, and more deformed adults than untreated larvae. The incorporation of methoxyfenozide into the diet had a signiϐicant effect on the timing of larval development. The development period for males and females was about seven days longer than the controls for both concentrations tested. In contrast, the compound affected neither pupae nor adult longevity. Finally, S. frugiperda adults that resulted from ϐifth instars treated with methoxyfenozide were not affected in their mean cumulative number of eggs laid per female (fecundity), nor percentages of eggs hatched (fertility), or the sex ratio. Our results suggest that the combination of lethal and sublethal effects of methoxyfenozide may have important implications for the population dynamics of the fall armyworm