Alteration of mitochondrial structure and heme biosynthetic parameters in liver and kidney cells by bismuth

Ultrastructural and biochemical studies were conducted to evaluate the effects of bismuth, a potentially toxic group V trace metal, on organelle structure and heme biosynthetic parameters in rat liver and kidney cells. Bismuth subnitrate (BiONO₃) was administered subcutaneously to male rats in 0, 20, 40, or 80 mg/kg doses 16 hr prior to euthanasia. Electron microscopy revealed swollen mitochondria and distortion of mitochondrial inner membranes in liver and renal proximal tubule cells at 40 and 80 mg/kg dose levels. In liver, dose-related decreases were observed in the activities of the mitochondrial enzymes, δ-aminolevulinic acid (ALA) synthetase and heme synthetase, and of the cytoplasmic enzyme, ALA dehydratase, to 51, 48, and 35% of levels seen in untreated controls, respectively. In kidney, ALA synthetase and ALA dehydratase, but not heme synthetase, were depressed in vivo to 32 and 20% of control, respectively. Studies in vitro conducted for 1-hr periods with Bi concentrations at 0, 0.1, 0.2, or 0.4 mm in reaction mixtures revealed that the direct action of the metal on membranal enzymes only partially accounts for the impairment of the activity of membranal enzymes. These studies demonstrate that the initial acute effects of bismuth in liver and kidney cells include distortion of mitochondrial membranes and direct inhibition of specific heme pathway enzymes. Both effects contribute to compromise of membrane-associated enzymatic functions. These findings are comparable to those previously reported of other trace metals with known toxicologic potential and may represent early events in bismuth-induced cell injury.     

The effects of ethanol,estrogen, and hexachlorobenzene on the activities of hepatic δ-aminolevulinate synthetase, δ-aminolevulinate dehydratase,and uroporphyrinogen decarboxylase in male rats

To determine if clinically observed disorders in heme biosynthetic enzymes, known as sporadic porphyria cutanea tarda (PCT), could be reproduced in experimental animals, male Fischer rats were treated with ethanol, estrogen and hexachlorobenzene (HCB). A series of heme biosynthetic enzymes were assayed. In the rats given free access to 8% ethanol-drinking water for 15 weeks, -aminolevulinate (ALA) dehydratase was significantly reduced in erythrocytes. In the liver, ALA synthetase and uroporphyrinogen (UROgen) decarboxylase activities remained unchanged. In bone marrow cells, these activities did not change markedly. In the rats treated with estrogen (1 mg estrioltripropionate /rat/week, IM), no body weight gain was observed during the treatment for 15 weeks and urinary ALA excretion increased to 1.7 fold over normal level. In the liver, a significant increase was observed in the activity of ALA dehydratase, but other enzymes remained within the normal level. In bone marrow cells and erythrocytes, ALA dehydratase was also increased. ALA synthetase increased only in bone marrow cells to 2.1 times higher than the control level. In rats fed 0.3% HCB-diet for 8 weeks, urinary excretion of ALA, coproporphyrin and uroporphyrin increased to 2.4, 3.3 and 3.8 times higher than the controls, respectively. In the liver, an increase was observed in ALA synthetase, while a decrease was observed in ALA dehydratase and UROgen decarboxylase. In bone marrow cells and erythrocytes, ALA dehydratase was reduced and activities of other enzymes did not show any changes.These results indicate that alcohol, estrogen and HCB do not produce phenomena similar to those observed clinically in PCT.     

The effect of lead on calcium release activated calcium influx in primary cultures of human osteoblast-like cells

To further understand the significance of bone as a target tissues of lead toxicity, as well as a reservoir of systemic lead, it is necessary to define the effect of lead on the calcium release activated calcium influx (CRACI) in primary cultures of human osteoblast-like cells (OLC). Pb²⁺ inhibited the immediate CRACI dose-dependent manner. Influx of Pb²⁺ into human OLC was increased dose-dependent manner. The present study demonstrates that the interference of Pb²⁺ with CRACI of human OLC is at least twofold: (1) the initiation of CRACI, i.e., the measurable influx of Ca²⁺ upon Ca²⁺ readdition, is partially inhibited by Pb²⁺ and (2) the influx of Pb²⁺ was enhanced after CRACI had been induced.     

Effects of lead on haem biosynthesis during erythroid differentiation in vitro

Murine erythroleukaemia cells (MELC) are erythroid precursor cells that undergo erythroid differentiation in the presence of the inducer hexamethylene bisacetamide (HMBA). The effects of lead on haem biosynthesis in MELC following HMBA-induced differentiation were studied. MELC were induced with HMBA in the presence of 20, 40 and 80 μ -lead acetate and cell density, haem content, incorporation of ¹⁴C-labelled δ-aminolaevulinic acid (ALA) into haem, and the activities of the enzymes δ-aminolaevulinic acid dehydratase (ALA-D), uroporphyrinogen I synthetase (URO-S) and ferrochelatase (FERRO) were determined. MELC exposed to 80 μ -lead showed significant erythroid hypoplasia (40–50%) and a significant decrease (30–50%) in haem content at 2, 4 and 6 days after induction in comparison with the controls. Significant inhibition of ALA-D, the most sensitive index, was noted at 20 μ -lead, and at 80 μ -lead ALA-D activity was decreased by 60–80% in comparison with the controls. URO-S and FERRO showed significant decreases of 34% and 50%, respectively, at 80 μ -lead. A decrease of 50% in the incorporation of [¹⁴C]ALA into haem at 80 μ -lead indicated an impairment in haem synthesis. The results suggest that the impairment of haem formation by lead is coincident with the production of severe erythroid hypoplasia.     

Rhizobium tibeticum activated with a mixture of flavonoids alleviates nickel toxicity in symbiosis with fenugreek (Trigonella foenum graecum L.)

The objective of this study is to explore the response of an activated Rhizobium tibeticum inoculum with a mixture of hesperetin (H) and apigenin (A) to improve the growth, nodulation, and nitrogen fixation of fenugreek (Trigonella foenum graecum L.) grown under nickel (Ni) stress. Three different sets of fenugreek seed treatments were conducted, in order to investigate the activated R. tibeticum pre-incubation effects on nodulation, nitrogen fixation and growth of fenugreek under Ni stress. Group (I): uninoculated seeds with R. tibeticum, group (II): inoculated seeds with uninduced R. tibeticum group (III): inoculated seeds with induced R. tibeticum. The present study revealed that Ni induced deleterious effects on rhizobial growth, nod gene expression, nodulation, phenylalanine ammonia-lyase (PAL) and glutamine synthetase activities, total flavonoids content and nitrogen fixation, while the inoculation with an activated R. tibeticum significantly improved these values compared with plants inoculated with uninduced R. tibeticum. PAL activity of roots plants inoculated with induced R. tibeticum and grown hydroponically at 75 and 100 mg L^?1 Ni and was significantly increased compared with plants receiving uninduced R. tibeticum. The total number and fresh mass of nodules, nitrogenase activity of plants inoculated with induced cells grown in soil treated up to 200 mg kg^?1 Ni were significantly increased compared with plants inoculated with uninduced cells. Plants inoculated with induced R. tibeticum dispalyed a significant increase in the dry mass compared with those treated with uninduced R. tibeticum. Activation of R. tibeticum inoculum with a mixture of hesperetin and apigenin has been proven to be practically important in enhancing nodule formation, nitrogen fixation and growth of fenugreek grown in Ni contaminated soils.     

Heavy metal modulation of lymphocyte activities. 1. In vitro effects of heavy metals on primary humoral immune responses

The ability of heavy metals to modulate in vitro primary humoral immune responses has been investigated. The relative immunosuppresive activities of the heavy metals tested were Hg²⁺ > Cu²⁺ > Mn²⁺ > Co²⁺; Cd²⁺ > Cr³⁺; Sn²⁺; Zn²⁺. Fe²⁺ had no significant effect on the development of SRBC-specific plaque-forming cells (PFC), and Pb²⁺ and Ni²⁺ enhanced the PFC response. The immunosuppressive activities of the heavy metals usually correlated with their toxicity and their inhibition of lymphocyte proliferation. The immunopotentiating metals, Pb²⁺ and Ni²⁺, alone induced lymphocyte proliferation, and they increased the proliferative response induced by LPS and 2-ME but not Con A and PHA. Although Pb²⁺ and Ni²⁺ were the only two heavy metals tested which enhanced lymphocyte reactivity, they did not appear to function via similar mechanisms. Pb²⁺ appeared to enhance the development of PFC by directly interacting with lymphocytes and altering their activity since lymphocyte preincubation with Pb²⁺ was sufficient for enhancement; whereas, lymphocyte preincubations with Ni²⁺ did not induce enhancement. Pb²⁺ did not appear to alter the immunogenicity of the antigens employed; however, pretreatment of SRBC with Ni²⁺ eliminated their ability to stimulate a SRBC-specific response in vitro. Inhibition of the PFC response by the chelator EGTA could be reversed by Ca²⁺ or Ni²⁺, but not by Cu²⁺, Hg²⁺, Pb²⁺, or Zn²⁺. The stimulatory activity of Pb²⁺ and Ni²⁺ could not be accounted for by significant alteration of the antigenicity of the lymphocytes, because heavy metal preincubated syngeneic cells were not stimulatory for untreated syngeneic cells. On the other hand, Pb²⁺ and Ni²⁺ did slightly enhance the mixed lymphocyte culture (MLC) response to allogeneic cells; Hg²⁺ inhibited the MLC response.     

Inhibition of delta-aminolevulinic acid synthetase by thioacetamide and thioacetamide-S-oxide.

Thiocetamide and one of its metabolites, thioacetamide-S-oxide, were shown to inhibit δ-aminolevulinic acid (ALA) synthetase when administered in vivo to adult male mice. Thioacetamide and thioacetamide-S-oxide also inhibited the 3,5-dicarboethoxy-1,4-dihydrocollidine (DDC)-mediated induction of ALA synthetase when given either 1hr prior to or 3 hr after the administration of DDC. The results of these studies also indicate that thioacetamide-S-oxide is generally a more potent inhibitor of ALA synthetase than thioacetamide.     

TRPC1 is involved in Ca influx and cytotoxicity following Pb exposure in human embryonic kidney cells

Lead (Pb²⁺) is a divalent heavy metal ion which causes severe damage to almost all life forms and is therefore considered a notorious toxicant. Exposure to Pb²⁺ is associated with poor cognitive development in children at relatively low levels that previously were thought to be safe. The mechanism through which Pb²⁺ enters cells, however, is unclear. Previous studies have showed that Ca²⁺ release-activated Ca²⁺ protein 1 (Orai1), a component of store-operated Ca²⁺ channels (SOCs), contributes to Pb²⁺ cellular entry. Canonical transient receptor potential (TRPC1) channel 1 is a transient receptor potential (TRP) channel which is sometimes referred to as a SOC. The present study was designed to investigate the role of TRPC1 in Pb²⁺ entry and toxicity in human embryonic kidney cells (HEK293). Additionally, changes in intracellular Ca²⁺ concentration were determined through Fluo-4 and Mag-fluo-4 fluorescent Ca²⁺ imaging. Following Pb²⁺ exposure, there was a dose-dependent decrease in cell viability. Overexpression of TRPC1 increased Pb²⁺-induced cell death, while knockdown of this channel attenuated cell death. There was increased entry of Pb²⁺, as measured by inductively coupled plasma mass spectrometry (ICP-MS), following overexpression of TRPC1. Conversely, knockdown of TRPC1 led to a decrease in Pb²⁺ influx. Down-regulation of STIM1 by RNA interference attenuated the Pb²⁺ influx, and transfection with a mutant STIM1, which could not gate TRPC1, had a similar effect. Co-transfection of mutant STIM1 and mutant TRPC1, which restore the electrostatic interaction between STIM1 and TRPC1, resumed Pb²⁺ entry in HEK293 cells. Down-regulation of TRPC1 by RNA interference decreased Ca²⁺ influx whilst its overexpression increased Ca²⁺ entry in HEK293 cells. These results suggest that TRPC1 is involved in the cytotoxicity and entry of Pb²⁺ through molecular interactions with STIM1 and subsequent Ca²⁺ influx in HEK293 cells.     

SUBUNIT-DEPENDENT ACTION OF LEAD ON NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS EXPRESSED IN XENOPUS OOCYTES

1. Pb²⁺ affects neuronal nicotinic acetylcholine receptors (nAChR) in NIE-115 neuroblastoma cells in a dual manner. At nanomolar concentrations a blockade is observed, while at submillimolar concentrations this blocking effect is reversed. 2. The Xenopus oocyte expression system was used to examine whether the dual effect of Pb²⁺ is related to a differential action on nAChR subtypes. Effects of Pb²⁺ were investigated in oocytes expressing nAChR after co-injection of a3 and β2 or α3 and β4 cDNA. 3. At 1–250 μmol/L, Pb²⁺ causes a 10–1000% increase of the response mediated by the α3β2 nAChR. 4. Pb²⁺ blocks ACh-induced inward currents mediated by α3β4 nAChR. The inhibitory potency of Pb²⁺ greatly varies between cells. In 50% of the cells concentrations ≤μmol/L Pb²⁺ blocked the nicotinic response by 31–93%. In the other cells even at higher concentrations Pb²⁺ caused only 0–65% inhibition. 5. These results show that Pb²⁺ may both potentiate and block nAChR, depending on the type of nAChR subunit expressed.     

Muscarinic cholinergic modulation of synaptic transmission and plasticity in rat hippocampus following chronic lead exposure

The cholinergic system is believed to be associated with learning and memory functions. Lead (Pb²⁺) is a well-known neurotoxic metal that causes irreversible damage to the central nervous system (CNS). To investigate whether Pb²⁺ interferes with cholinergic modulation, we examined the effects of carbachol (CCh), a muscarinic cholinergic agonist, on synaptic transmission and plasticity in the CA1 area of the hippocampus of developmentally Pb²⁺-exposed rats. The results showed that: (1) In both control and Pb²⁺-exposed rats, 0.1 μM CCh significantly enhanced tetanus-induced long-term potentiation (LTP), while 5 μM CCh induced a reversible depression of field excitatory postsynaptic potentials (fEPSPs). However, both the enhancement of LTP and depression of fEPSPs were significantly smaller in Pb²⁺-exposed rats than in controls, suggesting that the extent of the effect of CCh on the cholinergic system was depressed by Pb²⁺. (2) In Pb²⁺-exposed rats, the enhancement of LTP induced by 0.1 μM CCh was attenuated by pirenzepine, a M₁AChR antagonist, but was not affected by methoctramine tetrahydrochloride (M-105), a M_2/4AChR antagonist. The depression of fEPSPs induced by 5 μM CCh was reduced by either pirenzepine or M-105. (3) Furthermore, paired-pulse facilitation (PPF) was not affected by 0.1 μM CCh in control and Pb²⁺-exposed rats but was increased by 5 μM CCh in either group; the increase in PPF was less pronounced in Pb²⁺-treated when compared to control rats. These results suggested that cholinergic modulation could be impaired by Pb²⁺, and this kind of impairment might occur via different mAChR subtypes. Our study delineated the effects of Pb²⁺ on muscarinic modulation, and this might be one of the underlying mechanisms by which Pb²⁺ impairs learning and memory.     

Analysis of Pb2+ Entry into Cultured Astroglia

Astroglia serve as a presumptive lead (Pb) sink in the brain;therefore, this study examined Pb entry into cultured rat astrogliautilizing the Ca²⁺ fluorophore indo-1 as a tool for detectingPb²⁺ entry during acute exposure. The interactions of Pb²⁺ withindo-1 were analyzed by fluorescence spectrophotometry in acell-free system. The emission spectrum of Pb²⁺/indo-1 was substantiallydifferent from that of Ca²⁺/indo-1 due to suppression of indo-1fluorescence emission intensity. Next, we established the presenceof L-type Ca²⁺ channels in astroglial cultures and demonstratedthat Pb accumulation is enhanced under serum-free conditionsand by the application of Bay-K 8644. Because acute exposureis of less toxicologic relevance than repeated low-level exposure,we then examined Pb uptake in cultures treated for up to 1 weekwith Pb. AAS revealed that Pb accumulation was accompanied byan increase in total cellular [Ca]. In addition, differencesin basal indo-1 fluorescence levels and differences in responsivenessto ionomycin were observed. Ionomycin induced an increase inthe fluorescence ratio in untreated cells but cells treatedfor 1 day with Pb showed no response to ionomycin. However,cells treated for 3 and 7 days showed a partial response toionomycin. TPEN was used to evaluate the interactions of Pb²⁺with indo-1 and only cells treated for 7 days showed a responseto TPEN. Thus, the present study characterizes Pb²⁺ entry intoastroglia via L-type Ca²⁺ channels and presents the possibilityof using indo-1 for analysis of Pb²⁺ uptake and the subsequentneurotoxic events in astroglia.     

Bystander effects of PC12 cells treated with Pb depend on ROS-mitochondria-dependent apoptotic signaling via gap-junctional intercellular communication

The demonstration of bystander effect, which means injured cells propagate damage to neighboring cells, in whole organisms has clear implication of the potential relevance of the non-targeted response to human health. Here we show that 10 μM lead acetate, the optimum concentration for inducing apoptosis confirmed by the expression levels of Bax and Bcl-2, can also induce rat pheochromocytoma (PC12) cells to exert bystander effects to neighboring cells. In a novel co-culture system, GFP-PC12 (Pb²⁺) cells, which were stable transfected with EF1A-eGFP and pre-exposed with lead acetate, were co-cultured with unexposed PC12 cells at a 1:5 ratio. Parachute assays demonstrated the functional gap-junctional intercellular communication (GJIC) formed between Pb²⁺-exposed and unexposed cells. The Pb²⁺-exposed cells induced very similar effects on neighboring unexposed cells to apoptosis coincide with intracellular ROS generation and the collapse of mitochondrial membrane potential (Δψm). Furthermore, carbenoxolone (CBX), a blocker of GJIC, inhibited the bystander effects. The results indicate that the Pb²⁺-induced insults propagate through GJIC between PC12 cells, while inducing the bystander cells to apoptosis via ROS-mitochondria-dependent apoptotic signaling.     

Correlation of the physicochemical properties of metal ions with their activation and inhibition of human erythrocytic δ-aminolevulinic acid dehydratase (ALAD) in vitro

Using normal adult human whole blood hemolysates this study determined, in a dose-response fashion, the in vitro effects of Na⁺, Mg²⁺, Al³⁺, Mn²⁺, Cu²⁺, Ag⁺, Zn²⁺, Cd²⁺, Hg²⁺, Ga³⁺, In³⁺, Sn²⁺, Sn⁴⁺, and Pb²⁺ on normal erythrocytic ALAD. The effects of these 14 metal ions on erythrocytic ALAD 50% inhibited by Pb²⁺ were also determined as was the ability of a maximum stimulatory concentration of Zn²⁺ to prevent or reverse the effects of these metal ions on erythrocytic ALAD. The effects of these metal ions were then classified in terms of their oxidation state, characteristic coordination number, coordination geometry, and hard and soft Lewis acid characteristics in order to determine the physical and chemical properties associated with the ability of a metal ion to activate or inhibit erythrocytic ALAD and whether these properties are unique to a single metal ion. Preincubation studies established Zn²⁺ to be the only metal ion to both activate erythrocytic ALAD and to prevent or reverse the Pb²⁺-induced inhibition of erythrocytic ALAD in vitro even after prolonged contact with the enzyme. Therefore future investigations of the use of nontoxic salts of Zn²⁺ as a prophylactic agent or therapeutic adjunct in the prevention or treatment of lead poisoning with its possibly toxic accumulation of ALA are proposed.     

Effects of inorganic lead in vitro on ion exchanges and respiratory metabolism of rat kidney cortex

The effects of Pb²⁺ added in vitro to tissue slices, isolated tubules and isolated mitochondria of rat kidney cortex have been studied. Slices were depleted of K⁺ and loaded with Na⁺, Cl⁻ and water by pre-incubation at 1° C, and reversal of these changes was then induced by incubation under metabolically favourable conditions. The net reaccumulation of K⁺ was reduced by a maximum of 30% when Pb²⁺ was present in the medium, the maximal effect being caused by 200 μM Pb²⁺. Lead also caused a reduction of Na⁺ extrusion which was approximately equimolar with its effect on K⁺, but it did not affect the extrusion of Cl⁻ and water. The initial rates of the net, active movements of K⁺ and Na⁺ were not altered by Pb²⁺, divergence from control values only being noted after 15–30 min incubation. The O₂ consumption and the ATP content were 25–30% lower in slices incubated with 200 μM Pb⁺ than in control slices; the effect on ATP content was not observed until incubation had continued for 30 min. In tubules isolated from the renal cortex, the rate of respiration (50%) and ATP content (30%) were also partly reduced by 200 μM Pb²⁺. The consumption of O₂ by mitochondria isolated from the cortex was much more sensitive to Pb²⁺ added in vitro than the respiration of intact cells; the rate of respiration in state 3 (presence of phosphate acceptor) and the respiratory control ratio were drastically reduced, with half-maximal inhibition at 30 and 20 μM Pb²⁺ respectively. Comparison of the effects of Pb²⁺ on energy metabolism and ion transport of the slices with the corresponding effects of antimycin A and ouabain suggests that Pb²⁺ inhibited K⁺ and Na⁺ transport mainly as a consequence of a primary inhibition of the provision of ATP.     

Induction of benzo[a]pyrene metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin in primary cultures of adult rat hepatocytes

Primary hepatocyte cultures prepared from adult male Sprague-Dawley rats were extremely sensitive to induction of benzo[a]pyrene (BaP) metabolism by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), added to the cells in culture. A 48-hr exposure to 1 pmol TCDD/10⁶ cells resulted in a 4-fold induction of BaP metabolism. Significant induction was produced by a 48-hr exposure to a dose as low as 0.01 pmol (3 pg) TCDD/10⁶ cells. Cells exposed to TCDD from 7 to 55 hr in culture and then assayed for BaP metabolism responded to much lower doses of TCDD than did cells assayed at 36 hr in culture after being exposed to TCDD from 7 to 36 hr in culture. The doses of TCDD required for half-maximal induction of BaP metabolism were 24.8 and 164 fmol TCDD/10⁶ cells for cells assayed at 55 and 36 hr, respectively. Hepatocytes treated with TCDD starting at later times in culture showed a much more rapid time course of induction than did cells treated at earlier times in culture. A 2.6-fold induction occurred in cells treated from 49 to 55 hr in culture, compared to a 1.2-fold induction from 31 to 37 hr in culture. In contrast to cells exposed to TCDD from 7 to 55 hr in culture, cells exposed from 49 to 55 hr in culture were nearly as sensitive to inhibition of BaP metabolism by SKF 525-A as were uninduced control cells. An apparent derepression of the induction of BaP metabolism is occurring in primary hepatocyte cultures, resulting in an increase in the intitial rate of induction by TCDD and a decrease in the dose of TCDD required to obtain the half-maximum response.