A Kinetic Approach for Assessing the Uptake of Ag from Pristine and Sulfidized Ag Nanomaterials to Plants.

Nanomaterials (NMs) are thermodynamically unstable by nature, and exposure of soil organisms to NMs in the terrestrial environment cannot be assumed constant. Thus, steady-state conditions may not apply to NMs, and bioaccumulation modeling for uptake should follow a dynamic approach. The one-compartment model allows the uptake and elimination of a chemical to be determined, while also permitting changes in exposure and growth to be taken into account. The aim of the present study was to investigate the accumulation of Ag from different Ag NM types (20 nm Ag0 NMs, 50 nm Ag0 NMs, and 25 nm Ag2 S NMs) in the crop plant wheat (Triticum aestivum). Seeds were emerged in contaminated soils (3 or 10 mg Ag/kg dry soil, nominal) and plants grown for up to 42 d postemergence. Plant roots and shoots were collected after 1, 7, 14, 21, and 42 d postemergence; and total Ag was measured. Soil porewater Ag concentrations were also measured at each sampling time. Using the plant growth rates in the different treatments and the changing porewater concentrations as parameters, the one-compartment model was used to estimate the uptake and elimination of Ag from the plant tissues. The best fit of the model to the data included growth rate and porewater concentration decline, while showing elimination of Ag to be close to zero. Uptake was highest for Ag0 NMs, and size did not influence their uptake rates. Accumulation of Ag from Ag2 S NMs was lower, as reflected by the lower porewater concentrations. Environ Toxicol Chem 2021;40:1861-1872. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Extending standard testing period in honeybees to predict lifespan impacts of pesticides and heavy metals using dynamic energy budget modelling.

Concern over reported honeybee (Apis mellifera spp.) losses has highlighted chemical exposure as a risk. Current laboratory oral toxicity tests in A. mellifera spp. use short-term, maximum 96 hour, exposures which may not necessarily account for chronic and cumulative toxicity. Here, we use extended 240 hour (10 day) exposures to examine seven agrochemicals and trace environmental pollutant toxicities for adult honeybees. Data were used to parameterise a dynamic energy budget model (DEBtox) to further examine potential survival effects up to 30 day and 90 day summer and winter worker lifespans. Honeybees were most sensitive to insecticides (clothianidin > dimethoate ≫ tau-fluvalinate), then trace metals/metalloids (cadmium, arsenic), followed by the fungicide propiconazole and herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). LC50s calculated from DEBtox parameters indicated a 27 fold change comparing exposure from 48 to 720 hours (summer worker lifespan) for cadmium, as the most time-dependent chemical as driven by slow toxicokinetics. Clothianidin and dimethoate exhibited more rapid toxicokinetics with 48 to 720 hour LC50s changes of <4 fold. As effects from long-term exposure may exceed those measured in short-term tests, future regulatory tests should extend to 96 hours as standard, with extension to 240 hour exposures further improving realism.

Prevention of Ca(2+)-induced or thromboxane B2-induced hepatocyte plasma membrane bleb formation by thromboxane receptor antagonists.

Isolated hepatocytes incubated in the presence of either Ca2+ ionophore A23187 or thromboxane B2 develop many plasma membrane blebs which are a characteristic feature of toxic or ischaemic cell injury. When hepatocytes are incubated in the presence of both Ca2+ ionophore A23187 and any one of three thromboxane receptor antagonists (SK and F 88046, B.M. 13505, B.M. 13177), bleb formation is strongly inhibited. Hepatocytes incubated in the presence of both thromboxane B2 and any one of the three thromboxane receptor antagonists are also well protected from the formation of blebs. Treatment of isolated hepatocytes with Ca2+ ionophore A23187 is known to stimulate the production of thromboxanes. The data presented are consistent with thromboxane B2 acting as an intermediary in a proposed mechanism of cell injury and death in which elevated cytosolic free Ca2+ levels activate phospholipase A2 and the arachidonate cascade.

Prevention of thromboxane B2-induced hepatocyte plasma membrane bleb formation by certain prostaglandins and a proteinase inhibitor.

Isolated hepatocytes incubated in the presence of thromboxane B2 developed many plasma membrane blebs which are a characteristic feature of toxic or ischaemic cell injury. When hepatocytes were incubated in the presence of both thromboxane B2 and the non-lysosomal proteinase inhibitor, leupeptin, were also well protected from the formation of blebs. This implies that thromboxane B2 is able to activate non-lysosomal proteinases which appear to attack certain cytoskeletal proteins. The data presented are consistent with thromboxane B2 acting as an intermediary in a proposed mechanism of cell injury and death in which elevated cytosolic free Ca2+ levels activate phospholipase A2 and the arachidonic acid cascade.

Development of the cytosolic defence system against microsomal lipid peroxidation in rat liver.

Ascorbate-induced lipid peroxidation in rat liver microsomes reaches the adult level in 2-3 days. NADPH-induced peroxidation develops more gradually, in parallel with the activity of NADPH-cytochrome P-450 reductase, attaining adult levels by 10-12 days. The glutathione-dependent cytosolic enzyme activity which inhibits peroxidation is inhibited by bromosulphophthalein. The development of this system lags behind the development of microsomal lipid peroxidation between the ages of 2 and 20 days, allowing peroxidation to proceed.

Resistance to nitric oxide-mediated apoptosis in HL-60 variant cells is associated with increased activities of Cu,Zn-superoxide dismutase and catalase.

Nitric oxide (NO) released from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (DETA/NO or NOC-18) induces apoptosis in human leukemia HL-60 cells. In this study, we isolated a HL-60 variant cell line, HL-NR6, that is resistant to DETA/NO toxicity as assessed by DNA fragmentation, morphology, and colony forming ability. The variant cells also showed resistance to reactive oxygen species (ROS) such as superoxide and hydrogen peroxide as well as NO donors, but not to anti-tumor drugs. We found that HL-NR6 cells when compared with HL-60 cells possessed twice the activities of Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase, but no change in Mn-SOD nor in glutathione peroxidase. Immunoblotting confirmed the high levels of both enzymes in the variant cell. We also observed that ROS generation following DETA/NO exposure was substantially higher in HL-60 cells than in HL-NR6 cells, using the 2',7'-dichlorofluorescein fluorometric method. Moreover, the SOD mimetic Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin and exogenous catalase effectively attenuated DETA/NO-elicited DNA fragmentation in HL-60 cells. Taken together, these data suggested that the NO resistance in HL-NR6 cells is associated with the increased Cu,Zn-SOD/catalase and that NO-mediated apoptosis in HL-60 cells is correlated with the generation of ROS and derived molecules like peroxynitrite.

Developmental patterns of alcohol dehydrogenase and aldehyde dehydrogenases in homogenates and subcellular fractions of rat liver.

Both alcohol dehydrogenase (ADH) and the two isoenzymes of aldehyde dehydrogenase (ALDH-I-NAD+ and ALDH-II-NAD+) were first detected in foetal rat liver about 5 days before birth. All enzymes developed gradually and showed no abrupt increases in activity. The specific activities of ALDH-I-NAD+ and ALDH-II-NAD+ in the mitochondrial fractions, ALDH-II-NAD+ in the microsomal fractions and ADH in liver homogenates all produced a major percentage of the adult activity within a month, whereas the total activities increased over a longer part of the developmental period.

Decline in respiratory control ratio of rat liver mitochondria in old age.

Rat liver mitochondria isolated from old animals (27-33 months) showed a clear decline in the state 3 rate of respiration (presence of ADP) compared with mitochondria from mature animals (3-12 months) when using either succinate or NAD+-linked substrates. The state 4 rate of respiration (absence of ADP), which is a sensitive indicator of damage to the inner mitochondrial membrane, remained unchanged. Consequently the respiratory control ratio (state 3/state 4) declined. A loss of reserve capacity to maintain the ADP:O ratio was also observed. These observations indicate a decline in energy production in old animals.

Selective inhibition of formyl-methionyl-leucyl-phenylalanine (fMLF)-dependent superoxide generation in neutrophils by pravastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.

It has been shown previously that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, such as compactin, lovastatin, and pravastatin, block cholesterol synthesis, suppress lymphocyte functions, and beneficially affect atherogenesis. Recently, it was reported that compactin and lovastatin inhibit the respiratory burst of DMSO-differentiated HL-60 cells, an effect reversed by mevalonic acid. The mode of action of these inhibitors in this role is not understood fully. Thus, we studied the mechanism of inhibition of neutrophil superoxide (O2*-) generation by pravastatin and found that pravastatin at 0.5 mM inhibited the receptor-mediated tyrosine kinase (TK)-dependent pathway of O2*- generation and also luminol chemiluminescence but not the protein kinase C (PKC)-dependent or the TK- and PKC-independent pathways of O2*- generation in neutrophils. Pravastatin also inhibited the tumor necrosis factor-alpha- and formyl-methionyl-leucyl-phenylalanine-induced phosphorylation of a tyrosine of a 115-kDa protein. These effects were not reversed by mevalonate. From these results it is concluded that pravastatin inhibited receptor-mediated O2*-generation by decreasing tyrosine phosphorylation but not by inhibiting the formation of an intermediate in the biosynthesis of cholesterol.

Mechanism of apoptosis in HL-60 cells induced by n-3 and n-6 polyunsaturated fatty acids.

The biochemical properties and specificity of n-3 and n-6 polyunsaturated fatty acids (PUFAs) are not well known. Because PUFAs induce apoptosis of different cells, we studied the effect of various PUFAs, such as arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosapentaenoic acid (DPA), on the fate of cultured human promyelocytic leukemia cells (HL-60) to elucidate the mechanism of apoptosis and the difference in action between n-3 and n-6 PUFAs. Fairly low concentrations of PUFAs inhibited the growth of HL-60 cells and induced their apoptosis by a mechanism that is sensitive to DMSO, an antioxidant, and z-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk), a pan-caspase inhibitor. PUFAs stimulated the generation of reactive oxygen species (ROS) and activated various types of caspase-like proteases, such as caspase-3, -6, -8, and -9, but not caspase-1. In addition, PUFAs triggered the reaction leading to the cleavage of Bid, a death agonist member of the Bcl-2 family, and also released cytochrome c from mitochondria into the cytosol. PUFAs also decreased the mitochondrial membrane potential of intact HL-60 cells. All of these actions of n-3 PUFAs were stronger than those of AA, an n-6 PUFA, although the mechanism is not known. PUFAs stimulate swelling and membrane depolarization of isolated mitochondria in a cyclosporin A-sensitive manner. The results indicated that PUFA-induced apoptosis of HL-60 cells may be caused, in part, by direct action on the cells and by activation of the caspase cascade through cytochrome c release coupled with mitochondrial membrane depolarization.

Photoactivated inhibition of superoxide generation and protein kinase C activity in neutrophils by blepharismin, a protozoan photodynamically active pigment.

Blepharismin is an endogenous photosensitizing pigment found in the protozoan Blepharisma. This pigment inhibited the generation of superoxide anion (O2-.) in neutrophils not only via a diacylglycerol-induced protein kinase C (PKC)-dependent reaction but also by an arachidonate-induced PKC-independent reaction. The inhibition was light and concentration dependent for both reactions. Light-activated inhibition was strong at wavelengths between 520 and 570 nm but not above 610 nm. PKC activity in neutrophils and from rat brain was inhibited by blepharismin in a light- and concentration-dependent manner. Moreover, arachidonate-activated NADPH oxidase activity in a cell-free system was also inhibited by the pigment in a light- and concentration-dependent manner. These results suggest that blepharismin inhibits NADPH oxidase activation through the non-specific inhibition of various membrane-bound enzymes and that this inhibition may also be correlated with that of PKC.

Effect of the antiretroviral agent hypericin on rat liver mitochondria.

The photosensitizing effect of hypericin (HY), an antiretroviral agent, on the functions of isolated rat liver mitochondria has been investigated. The respiratory control ratio (RCR), ADP/O and membrane potential of mitochondria were decreased by HY in a light-dependent manner. Uncoupled respiration of mitochondria in the presence of succinate was also inhibited by HY in a light-dependent manner. The ID50 of hypericin for these inhibitions was approximately 0.5 microM. These inhibitory effects of HY were not observed when photosensitization was conducted under anaerobic conditions and were not affected by desferrioxamine (DSF) or superoxide dismutase (SOD). Upon photosensitization of HY, mitochondria consumed oxygen in the absence of respiratory substrate with concomitant formation of thiobarbituric acid reactive substance (TBARS). The amount of oxygen consumed was 100-times greater than that of TBARS formed. The oxygen uptake was partially inhibited by NaN3, and formation of TBARS was inhibited by DSF. Upon photosensitization of HY in the presence of mitochondrial membranes, the electron spin resonance (ESR) signal of 2,2-dimethyl-5-hydroxy-1-pyrrolidinyloxyl (DMPO/.OH) was increased by a mechanism which was suppressed by DSF. An ESR signal for singlet oxygen bound to 2,5-dimethylfuran, 2,2,6,6-tetramethyl-4-piperidone (TEMP) was also detected under light in the presence of mitochondria. This signal of the TEMP-N-oxyl radical (TEMPO) was decreased by azide, which physically quenches singlet oxygen, but was increased by DSF. These results indicate that HY might inhibit mitochondrial functions by a type II photodynamic mechanism but that lipid peroxidation of biological membranes through an active oxygen-mediated photodynamic mechanism is not involved.

Caspase activation and cytochrome c release during HL-60 cell apoptosis induced by a nitric oxide donor.

Nitric oxide (NO) from (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1,2-diolate (NOC-18) induces apoptosis in human leukemia HL-60 cells. This effect was prevented by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), thereby implicating caspase activity in the process. NOC-18 treatment resulted in the activation of several caspases including caspase-3, -6, -8, and -9(-like) activities and the degradation of several caspase substrates such as nuclear lamins and SP120 (hnRNP-U/SAF-A). Moreover, release of cytochrome c from mitochondria was also observed during NOC-18-induced apoptosis. This change was substantially prevented by Z-VAD-FMK, thereby suggesting that the released cytochrome c might function not only as an initiator but also as an amplifier of the caspase cascade. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to NOC-18, supporting the above notion. Thus, NO-mediated apoptosis in HL-60 cells involves a caspase/cytochrome c-dependent mechanism.

Inhibition of stimulus-specific neutrophil superoxide generation by alpha-tocopherol.

Alpha-tocopherol but not 2-carboxy-2,5,7,8-tetramethyl-6-chromanol (trolox or CTMC) and 2,2,5,7,8 pentamethyl-6-hydroxy chromane (PMC), derivatives of alpha-tocopherol, inhibited the superoxide (O2-.) generation of rat peritoneal neutrophils (RPMN) induced by phorbol 12-myrisate 13-acetate (PMA). ID50 for neutrophils obtained from the peritoneal cavity of rat and guinea pig was about 1microM. This concentration, however, was much lower than that for the inhibition of PMA-activated phospholipid-dependent protein kinase (PKC) (ID50 = 30 microM). The alpha-tocopherol sensitive O2-. generation was also observed in neutrophils induced by dioctanoylglycerol (diC8) and calcium ionophore A23187 but not by formylmethionyl-leucyl-phenylalanine (FMLP), opsonized zymosan (OZ) and sodium dodecyl sulfate (SDS). The pattern of inhibition by alpha-tocopherol was quite similar to that of staurosporine, a specific inhibitor of PKC. The alpha-tocopherol content of RPMN was 12 ng/10(6) cells and a linear increase to 200 ng/10(6) cells by addition of alpha-tocopherol to the cell suspension corresponded with an increased inhibition of O2-. generation. These results indicate that both the chemical structure and the content of alpha-tocopherol might be important factors in O2-. generation by neutrophils.

Molecular mechanisms of apoptosis in HL-60 cells induced by a nitric oxide-releasing compound.

Nitric oxide (NO) generated from 1-hydroxy-2-oxo-3, 3-bis(2-aminoethyl)-1-triazene (NOC 18), an NO-releasing compound, induced monocytic differentiation of human promyelocytic leukemia HL-60 cells as assessed by expression of nonspecific esterases and morphologic maturation. Simultaneously, DNA fragmentation and morphological alterations typical of apoptosis were also induced. To investigate the mechanisms of apoptosis during differentiation of HL-60 cells induced by NO, the endogenous levels of Bcl-2 and Bax were assessed by immunoblotting. Treatment of cells with NOC 18 slightly reduced the level of Bcl-2 followed by Bax. These changes might be involved in the induction of apoptosis. The involvement of the activation of the interleukin-1 beta converting enzyme (ICE) family of proteases (caspases), such as ICE and CPP32, in the pathways was also investigated. CPP32, but not ICE, was strongly activated in response to NOC 18 stimulation, thereby implicating CPP32-like activity in the induction of apoptosis. Moreover, the possible involvement of tyrosine phosphorylation in apoptosis was investigated. Pretreatment of cells with herbimycin A, an inhibitor of tyrosine kinases, suppressed DNA fragmentation and CPP32-like activity, whereas pretreatment with vanadate, an inhibitor of tyrosine phosphatases, enhanced both parameters, suggesting that tyrosine phosphorylation might be involved in the pathways of apoptosis in HL-60 cells induced by NO.

Inhibition of neutrophil-superoxide generation by alpha-tocopherol and coenzyme Q.

Effects of various derivatives of alpha-tocopherol (VE) and coenzyme Q (CoQ) on superoxide (O2.-) generation of neutrophils and protein kinase C (PKC) activity were examined. VE and CoQ8 inhibited O2.- generation of neutrophils stimulated by a protein kinase C mediated process monitored by cytochrome c reduction and spin trapping methods. The inhibitory action was observed not only with alpha-tocopherol, but also with beta-, gamma-, delta-tocopherols and with tocol which is a chemical similar to VE but lacking methyl groups on the chromanol ring structure and which is not a radical scavenger. By contrast, no inhibition was observed with 2-carboxy-2, 5, 7, 8-tetramethyl-6-chromanol (CTMC, trolox) or 2, 2, 5, 7, 8,-pentamethyl-6-chromanol (PMC) which are water soluble VE derivatives having radical scavenging activity. Compounds having a similar isoprenoid chain, such as CoQ, also have inhibitory activity on PKC-dependent O2.- generation of neutrophils. The inhibitory activity of CoQ derivatives is dependent on the length of the unsaturated isoprenoid chain. CoQ derivatives having 16, 24 and 32 carbon isoprenoid chains corresponding to CoQ4, 6, and 8 inhibited O2.- generation but 4 and 40 carbon isoprenoid chains corresponding to CoQ2 and 10 had no inhibitory activity on O2.- generation. Alpha-tocopherol and CoQ inhibited PKC activity but the ID50 for O2.- generation and PKC activity was different for each compound. However, no direct relationship between VE content and O2.- generation of neutrophils was observed. These results suggest that isoprenoids of VE and CoQ participate in the inhibition of the NADPH oxidase activation system through modulation of the neutrophil membrane probably by the inhibition of PKC.

Studies on paracetamol-induced lipid peroxidation.

Post-mitochondrial supernatants isolated from livers of rats given a single large oral dose of paracetamol (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. As a result of paracetamol administration the level of reduced glutathione (GSH) declined to approx. 20-25% of the peak physiological value. Addition of reduced GSH to the supernatant inhibited the peroxidation. Paracetamol-induced lipid peroxidation was inhibited in vitro by antioxidants (e.g. vitamin E) but was unaffected by superoxide dismutase and mannitol. N-acetyl cysteine and cysteamine inhibited lipid peroxidation in vitro in a cytosol-dependent manner in the absence of glutathione. Lipid peroxidation probably occurs simultaneously with the proposed covalent binding of the active metabolite of paracetamol. Since the former process is known to cause severe and extensive membrane damage, it may be a very important factor in paracetamol-induced liver necrosis.

Histochemical and biochemical observations on the cytotoxicity of paracetamol and its effects on glycogen metabolism in rat liver.

The effects of paracetamol overdose on glycogen metabolism in rat liver have been investigated and related to its cytotoxicity. Paracetamol was administered to male rats by gavaging after a 24-h fast and refeeding was not permitted. An early (9-12-h) increase in histochemically demonstrable glycogen phosphorylase alpha activity in perivenous hepatocytes preceded major loss of membrane integrity as assessed by serum glutamate-pyruvate transaminase (SGPT) activity and uptake of trypan blue during perfusion. These changes occurred only after a decrease in the concentration of reduced glutathione, which is generally observed about 4 h after paracetamol treatment. The activation of glycogen phosphorylase in perivenous hepatocytes occurred concurrently with an increase in glycogen content of periportal hepatocytes, indicating a clear heterogeneity in the response of the two-cell populations to the hepatotoxin. The use of trypan blue perfusion together with histochemical techniques allowed changes in glycogen content and phosphorylase alpha activity of individual hepatocytes to be assessed with reference to the extent of membrane damage evident. The relevance of the results to possible mechanisms of hepatotoxicity is discussed.

Effect of old age on paracetamol-induced lipid peroxidation in rat liver.

Post-mitochondrial supernatants isolated from the livers of mature rats (3 to 6 months old) 2 h or more after the administration of a single large oral dose of paracetamol (800 mg/kg) showed rapid rates of lipid peroxidation when incubated in vitro. In similar experiments with old rats (27-30 months old) the time between administration of paracetamol and the onset of lipid peroxidation was much longer (6 h or more). In both age groups, lipid peroxidation was dependent on the depletion of glutathione from the liver.

Superoxide dismutase-like activity of metal substituted lactoferrin derivatives.

Lactoferrin (LF) is an iron-binding glycoprotein present in various secretions including milk and the specific granules of neutrophils. The main biological properties of this protein are thought to concern the regulation of iron absorption, antimicrobial activity and modulation of neutrophil activity. Copper bound LF (Cu-LF) inhibited the stimulation-dependent reduction of cytochrome c (Cyt. c) in guinea pig peritoneal neutrophils (GPMN) but were without effect on NADPH oxidase activity of the respiratory burst. However, Cu-LF stimulated the stimulation-dependent production of hydrogen peroxide as seen with superoxide dismutase (SOD). Similar but weaker inhibition of Cyt. c reduction than that shown by Cu-LF was observed with manganese-LF (Mn-LF) but not with ferrous-LF (Fe-LF) or apo-LF (Apo-LF). The inhibitory activity was concentration-dependent and the ID50s of Cu-LF and of Mn-LF were 0.1 and 5 microM, respectively. Reactive oxygen species (ROS) detected by luminol chemiluminescence (LCL) of stimulated-GPMN were partially inhibited by Cu-LF. Changes in LCL of stimulated GPMN induced by Cu-LF were similar to those of superoxide dismutase (SOD). Thus, it is concluded that low concentrations of Cu-LF had SOD-like activity and high concentrations of Cu-LF inhibited the stimulation-dependent generation of ROS.