Search

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C–40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

Peripheral arterial disease (PAD) has been associated with suboptimal treatment, high mortality, and high amputation rates. It is unclear how the COVID-19 (coronavirus disease 2019) pandemic affected this development in a long term context. This is a registry based, retrospective, nationwide cohort study including patients hospitalised with PAD as a main or secondary diagnosis and amputation surgery between 2012 – 2021 in Germany. Primary endpoints were population wide major and minor amputation rates, in hospital death, and in hospital mortality rates. Secondary endpoints were same admission revascularisations and in hospital death in the event of complications, i.e., failure to rescue (FTR). Pre-pandemic and pandemic trends, focusing on lockdown periods, were analysed. A total of 365 926 patient records with PAD and amputation surgery were analysed. The median patient age was 75 years and 28.8% were female. Overall population wide amputation and in hospital mortality rates (monthly decrease –0.002/100 000, p <.001, and –0.001/100 000, p <.001, respectively) and in hospital mortality rate (8.0% for 2012 – 2014 vs. 6.5% for 2020 – 2021; p <.001) declined between 2012 and 2020. Concurrently, same admission revascularisations increased (41.0% for 2012 – 2014 vs. 47.0% for 2020 – 2021; p <.001), while FTR decreased in a subset of complications (acute ischaemia, major bleeding, compartment syndrome, and mesenterial ischaemia). In the first pandemic lockdown, there was a temporary trend change to higher major amputations rates (+0.02/100 000; p <.001) and higher in hospital mortality rates (+0.007/100 000; p <.001), which changed to a decrease as of the second lockdown (–0.03/100 000, p =.034, and –0.010/100 000, p <.001, respectively) in an interrupted time series analysis. There was no statistically significant change in observed amputation rates during lockdowns, while observed in hospital mortality rates decreased by 12.0% in the first lockdown (0.22/100 000 vs. 0.25/100 000; p =.005) compared with reference periods of the two previous years. Between 2012 and 2021, pre-pandemic trends toward decreasing population wide overall amputation rates, fewer major amputations, more amputation related revascularisation procedures, and lower in hospital mortality were maintained despite a temporary trend to increased major amputations and in hospital mortality during the first COVID-19 related lockdown in Germany. [ABSTRACT FROM AUTHOR]

Certain ATP binding cassette (ABC) transporter proteins, such as zebrafish Abcb4, are efflux pumps acting as a cellular defence against a wide range of different, potentially toxic chemical compounds thus mediating so called multixenobiotic resistance (MXR). Certain chemicals target MXR proteins and, as so called chemosensitisers, inhibit the activity of these proteins thus increasing the toxicity of other chemicals that would normally be effluxed. In this study 14 pharmaceuticals and personal care products (PPCPs) that are being increasingly detected in aquatic systems, were assessed for interference with the MXR system of zebrafish ( Danio rerio ). Concentration dependent effects of test compounds were recorded with the dye accumulation assay using zebrafish embryos and in ATPase assays with recombinant zebrafish Abcb4. In the dye accumulation assay embryos at 24 h post fertilisation (hpf) were exposed to 8 µm rhodamine 123 along with test compounds for 2 h. The rhodamine 123 tissue levels upon the exposure served as a measure for MXR transporter efflux activity of the embryo (low rhodamine levels – high activity; high levels - low activity). The known ABC protein inhibitors MK571, vinblastine and verapamil served as positive controls. All tested PPCPs affected rhodamine 123 accumulation in embryos. For seven compounds rhodamine tissue levels were either both decreased and increased depending on the compound concentration indicating both stimulation and inhibition of rhodamine 123 efflux by those compounds, only increased (inhibition, six compounds) or only decreased (stimulation, one compound). Recombinant zebrafish Abcb4 was obtained with the baculovirus expression system and PPCPs were tested for stimulation/inhibition of basal transporter ATPase activity and for inhibition of the transporter ATPase activity stimulated with verapamil. Eight of the tested PPCPs showed effects on Abcb4 ATPase activity indicating that their effects in the dye accumulation assay may have indeed resulted from interference with Abcb4-mediated rhodamine 123 efflux. Slight stimulatory effects were found for musk xylene, nerol, isoeugenol, α-amylcinnamaldehyde, α-hexylcinnamaldehyde and simvastatin indicating Abcb4 substrate/competitive inhibitor properties of those compounds. Likewise, decreases of the verapamil-stimulated Abcb4 ATPase activity by diclofenac and fluoxetine may indicate competitive transporter inhibition. Sertraline inhibited the basal and verapamil-stimulated Abcb4 ATPase activities suggesting its property as non-competitive Abcb4 inhibitor. Taken together, our finding that chemically diverse PPCPs interfere with MXR efflux activity of zebrafish indicates that (1) efflux transporters may influence bioaccumulation of many PPCPs in fish and that (2) many PPCPs may act as chemosensitisers. Furthermore, it appears that interference of PPCPs with efflux activity in zebrafish embryos is not only from effects on Abcb4 but also on other efflux transporter subtypes. [ABSTRACT FROM AUTHOR]