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Benthic marine organisms such as mollusks are often exposed to periodic oxygen deficiency (due to the tidal exposure and/or seasonal expansion of the oxygen-deficient dead zones) and pollution by metals [e.g., cadmium, (Cd)]. These stressors can strongly affect mollusks' survival; however, physiological mechanisms of their combined effects are not fully understood. We studied the effects of Cd exposure on metabolic responses to prolonged anoxia and subsequent recovery in anoxia-tolerant intertidal mollusks Crassostrea virginica (eastern oysters). Anoxia led to an onset of anaerobiosis indicated by accumulation of L-alanine, acetate, and succinate. Prolonged anoxia (for 6 days) caused a decline in the maximum activity of electron transport chain and ADP-stimulated (state 3) oxygen uptake by mitochondria (M[O.sub.2]), but no change in the resting (state 4) M[O.sub.2] of oyster mitochondria, along with a slight but significant reduction of mitochondrial respiratory control ratio. During reoxygenation, there was a significant overshoot of mitochondrial M[O.sub.2] (by up to 70% above the normoxic steady-state values) in control oysters. Mild mitochondrial uncoupling during prolonged shutdown in anoxic tissues and a subsequent strong stimulation of mitochondrial flux during recovery may help to rapidly restore redox status and protect against elevated reactive oxygen species formation in oysters. Exposure to Cd inhibits anaerobic metabolism, abolishes reoxygenation-induced stimulation of mitochondrial M[O.sub.2], and leads to oxidative stress (indicated by accumulation of DNA lesions) and a loss of mitochondrial capacity during postanoxic recovery. This may result in increased sensitivity to intermittent hypoxia and anoxia in Cd-exposed mollusks and will have implications for their survival in polluted estuaries and coastal zones. air exposure; recovery; mitochondrial function; oxidative damage; mollusks doi: 10.1152/ajpregu.00324.2009.

Marine ectotherms, including oysters are exposed to variable environmental conditions in coastal shallow waters and estuaries. In the light of global climate change, additional stressors like pollution might pose higher risk to populations. On the basis of the concept of oxygen- and capacity-limited thermal tolerance in aquatic ectotherms (40), we show that a persistent pollutant, cadmium, can have detrimental effects on oysters (Crassostrea virginica). During acute warming from 20 to 28[degrees]C (4[degrees]C/48 h) standard metabolic rate (SMR) rose in control and cadmium-exposed (50 [micro]g [Cd.sup.2+]/1) animals, with a consistently higher SMR in Cd-exposed oysters. Additionally, Cd-exposed oysters showed a stronger temperature-dependent decrease in hemolymph oxygen partial pressures. This observation indicates that the effect of temperature on aerobic metabolism was exacerbated due to the additional Cd stress. The oxygen delivery systems could not provide enough oxygen to cover Cd-induced elevated metabolic demands at high temperatures. Interestingly, cardiac performance (measured as the heart rate and hemolymph supply to tissues) rose to a similar extent in control and Cd-exposed oysters with warming indicating that cardiac output was unable to compensate for elevated energy demand in Cd-exposed oysters. Together with the literature data on metal-induced reduction of ventilatory capacity, these findings suggest that synergistic effects of elevated temperatures and cadmium exposure led to oxygen limitation by impaired performance in oxygen supply through ventilation and circulation. Overall, cadmium exposure resulted in progressive hypoxemia in oysters at high temperatures, suggesting that the thermal tolerance window is narrowed in marine ectotherms inhabiting polluted areas compared with pristine environments. aerobic metabolism; cardiac performance; magnetic resonance imaging, hemolymph [Po.sub.2], anaerobiosis

In the adult rat, chronic cadmium intoxication induces nephropathy with Fanconi-like features. This result raises the question of whether intoxication of pregnant rats has any deleterious effects on renal function in their offspring. To test this hypothesis, we measured the renal function of 2- to 60-day-old postnatal offspring from female rats administered cadmium chloride by the oral route (0.5 rag. [kg.sup.-1] [day.sup.-1) throughout their entire gestation. Investigations of rat offspring from contaminated pregnant rats showed the presence of cadmium in the kidney at gestational day 20. After birth, the cadmium kidney concentration increased from postnatal day 2 to clay 60 (PND2 to PND60), presumably because of 1) milk contamination and 2) neonatal liver cadmium content release. Although the renal parameters (glomerular filtration, U/P inulin, and urinary excretion rate) were not significantly affected until PND45, renal failure appeared at PND60, as demonstrated by a dramatic decrease of the glomerular filtration rate associated with increased excretion of the main ions. In parallel, an immunofluorescence study of tight-junction protein expression of PND60 offspring from contaminated rats showed a disorganization of the tight-junction proteins claudin-2 and claudin-5, specifically expressed in the proximal tubule and glomerulus, respectively. In contrast, expression of a distal claudin protein, claudin-3, was not affected. In conclusion, in utero exposure of cadmium leads to toxic renal effects in adult offspring. These results suggest that contamination of pregnant rats is a serious and critical hazard for renal function of their offspring. heavy metals; renal failure; newborn; claudins

The nephrotoxic metal cadmium at micromolar concentrations induces apoptosis of rat kidney proximal tubule (PT) cells within 3-6 h of exposure. The underlying cell death pathways remain poorly defined. Using Hoechst 33342/ ethidium bromide nuclear staining and 3-(4,5-dimethyl-2-thiazolyl) 2,5-diphenyl-2H-tetrazolium bromide (MTT) cell death assays, 10-50 [micro]M cadmium induced apoptosis of immortalized rat kidney cells derived from the S1-segment of PT at 6 and 24 h, but necrosis at 24 h only. Cadmium (10-50 [micro]M) also caused mitochondrial cytochrome c (cyt. c)- and apoptosis-inducing factor release at 24 h, but not at 6 h, as measured by immunofluorescence imaging and immunoblotting. Caspases-9 and -3 were activated only by l0 [micro]M cadmium for 24 h, and accordingly apoptosis was significantly reduced by the respective inhibitors (z-LEHD-fmk, z-DEVD-fmk; 10 [micro]g/ml) at 24 h, but not at 6 h, without affecting necrosis. At 6 h, 10 [micro]M cadmium increased the activity of the calcium-activated protease calpain, but not at 24 h, and calpain inhibitors (ALLN, PD 150606; 10-30 [micro]M) blocked apoptosis by 10 [micro]M cadmium at 3-6 h. However, PD-150606 also attenuated caspase-3 activity and apoptosis at 24 h, suggesting calpain-dependent caspase activation. Thus cadmium-induced apoptosis of PT cells involves a complex and sensitive interplay of signaling cascades involving mitochondrial proapoptotic factors, calpains and caspases, whose activation is also determined by cadmium concentration and the duration of cadmium exposure. heavy metals; apoptosis-inducing factor; cytochrome c; cysteine proteases

The environmental pollutant cadmium affects human health, with the kidney being a primary target. In addition to proximal tubules, glomeruli and their contractile mesangial cells have also been identified as targets of cadmium nephrotoxicity. Glomerular contraction is thought to contribute to reduced glomerular filtration, a characteristic of cadmium nephrotoxicity. Because p38 MAPK/HSP25 signaling has been implicated in smooth muscle contraction, we examined its role in cadmium-induced contraction of mesangial cells. We report that exposure of mesangial cells to cadmium resulted in 1) cell contraction, 2) activation of MAP kinases, 3) increased HSP25 phosphorylation coincident with p38 MAP kinase activation, 4) sequential phosphorylation of the two phosphorylation sites of mouse HSP25 with Ser15 being phosphorylated before Ser86, 5) reduction of oligomeric size of HSP25, and 6) association of HSP25 with microfilaments. Exposure of isolated rat glomeruli to cadmium also resulted in contraction and increased HSP25 phosphorylation. The cadmium-induced responses were inhibited by the specific p38 MAP kinase inhibitor SB-203580, and cadmium-induced phosphorylation of HSP25 was inhibited by expression of a dominant-negative p38 MAP kinase mutant. These findings tentatively suggest that cadmium-induced nephrotoxicity results, in part, from glomerular contraction due to p38 MAP kinase/ HSP25 signaling-dependent contraction of mesangial cells. With regard to the cellular action of HSP25, these data support a change in paradigm: in addition to its weII-established cytoprotective function, HSP25 may also be involved in processes that ultimately lead to adverse effects, as is observed in the response of mesangial cells to cadmium. cadmium nephrotoxicity; actin; microfilaments; phosphorylation

DMT1 (divalent metal transporter 1) is a hydrogen-coupled divalent metal transporter with a substrate preference for iron, although the protein when expressed in frog oocytes transports a broad range of metals, including the toxic metals cadmium and lead. Wild-type Caco-2 cells displayed saturable transport of lead and iron that was stimulated by acid. Cadmium and manganese inhibited transport of iron, but zinc and lead did not. The involvement of DMT1 in the transport of toxic metals was examined by establishing clonal DMT1 knockdown and control Caco-2 cell lines. Knockdown cell lines displayed much lower levels of DMT1 mRNA and a smaller [V.sub.max] for iron uptake compared with control cell lines. One clone was further characterized and found to display an ~50% reduction in uptake of iron across a pH range from 5.5 to 7.4. Uptake for cadmium also decreased 50% across the same pH range, but uptake for lead did not. These results show that DMT1 is important in iron and cadmium transport in Caco-2 cells but that lead enters these cells through an independent hydrogen-driven mechanism. divalent metal transporter 1; transport; intestine

The toxicity of cadmium on renal cells was investigated using the LLC-PK1 and the Madin-Darby canine kidney cell lines as in vitro models of proximal and distal segments of the nephron respectively. Results reveal the cadmium decreases ATP levels, interferes with the cadherin-cathenin complex and even the tight junction between cells. The effects of cadmium are more pronounced on the LLC-PK1 cell line.

The effects of preincubation with cadmium on calcium uptake and cadmium accumulation in tilapia larvae were investigated. Results reveal that cadmium preexposure promotes some form of adaptation in tilapia larvae that lessens the deleterious effects of cadmium. Further, larvae preincubated with cadmium are more efficient in calcium uptake. The total amount of accumulated cadmium does not affect survival as much as the interference of cadmium on calcium metabolism.

The effects of external Cd2+ on Na/Ca exchange and external L-type Ca channel current (ICa,L) were analyzed in rabbit ventricular myocytes via whole patch-clamp technique. The Na/Ca exchange and ICa,L of whole cell patch-clamped rabbit myocytes were inhibited by external Cd2+ in a dose-dependent manner. Furthermore, the effects of Cd2+ on Na/Ca exchange were significant during the first ramp depolarization of the myocytes and they did not increase with prolonged application of Cd2+.

The sensitivity of the Purkinje-ventricular muscle junctions (PVJ) was compared with the sensitivity of the Purkinje cells in the canine papillary muscles to modulators of L-type calcium channels. L-type calcium channel modulation with cadmium and isoproterenol produced greatly affected the regions with discontinuous conductions compared to regions with continuous conduction. Furthermore, cadmium produced a significant record delay on PVJ compared to its effects on Purkinje.

Experimental studies of the role of cadmium chloride (CdCl2) in the connective tissue lung diseases show that CdCl2 suppresses the generation of fibroblast procollagen and the growth of fibroblasts but has no effect on the synthesis of noncollagen proteins. The inhibitory action is dose-dependent, with maximum inhibition occurring at 40 micro M and 10 micro M of CdCl2 in human and rat fetal fibroblasts. The reduction in procollagen synthesis and the rise in the degradation of the newly synthesized procollagen inhibit procollagen production, indicating that CdCl2 contributes to tissue pathogenesis in the lungs.

The involvement of iron (Fe) transporters in the uptake of cadmium (Cd) was examined in Madin-Darby kidney cells (MDCK). The uptake of Cd displayed properties that are associated with the Fe transporter divalent metal transporter 1 (DMT1). For example, the uptake of Cd and Fe was reduced by altering the cell membrane potential. The uptake of Cd was blocked by Fe, and the uptake of Fe was blocked by Cd. Also, the uptake of Cd and Fe was higher in MDCK cells bathed in a buffer at low pH. Increased uptake of Fe and Cd was observed in the HEK-293 cell line overexpressing DMT1. Overnight treatment of MDCK cells with the protein kinase C activator phorbol 12,13-dibutyrate (PDBu) resulted in increased uptake of Cd and Fe and an increase in DMT1 mRNA. An increase in newly transcribed DMT1 mRNA was not observed, suggesting that PDBu does not increase DMT1 mRNA by activating transcription. Rather, the increase was most likely due to greater stability of DMT1 mRNA, because the rate of degradation of DMT1 mRNA was slower in MDCK cells treated with PDBu. Our results suggest that Fe and Cd are transported in MDCK cells by a transporter with biochemical properties similar to those of DMT1. divalent metal transporter 1; cadmium; Madin-Darby canine kidney cells

Ma, Rong, Sonja Smith, Angie Child, Pamela K. Carmines, and Steven C. Sansom. Store-operated [Ca.sup.2+] channels in human glomerular mesangial cells. Am J Physiol Renal Physiol 278: F954-F961, 2000.--Experiments were performed to identify the biophysical properties of store-operated [Ca.sup.2+] channels (SOC) in cultured human glomerular mesangial cells (MC). A fluorometric technique (fura 2) was utilized to monitor the change in intracellular calcium concentration ([[[Ca.sup.2+]].sub.i]) evoked by elevating external [[Ca.sup.2+]] from 10 nM to 1 mM ([Delta][[Ca.sup.2+]]). Under control conditions, [Delta][[Ca.sup.2+]] averaged 6 nM and was unaffected by elevating bath [[K.sup.+]]. After treatment with 1 [micro]m thapsigargin to deplete the intracellular [Ca.sup.2+] store, the change in [[[Ca.sup.2+]].sub.i] ([Delta][[[Ca.sup.2+]].sub.th]) averaged 147 [+ or -] 16 nM. In thapsigargin-treated MC studied under depolarizing conditions (75 mM bath [K.sup.+]), [Delta][[[Ca.sup.2+]].sub.th] was 45 [+ or -] 7 nM. The [Delta][[[Ca.sup.2+]].sub.th] response of thapsigargin-treated cells was inhibited by [La.sup.3+] ([IC.sub.50] = 335 nM) but was unaffected by 5 [micro]m [Cd.sup.2+]. In patch clamp studies, inward currents were observed in cell-attached patches with either 90 mM [Ba.sup.2+] or [Ca.sup.2+] in the pipette and 140 mM KCl in the bathing solution. The single-channel conductance was 2.1 pS with [Ba.sup.2+] and 0.7 pS with [Ca.sup.2+]. The estimated selectivities were [Ca.sup.2+] [is greater than] [Ba.sup.2+] [much greater than] [K.sup.+]. These channels were sensitive to 2 [micro]m [La.sup.3+], insensitive to 5 [micro]M [Cd.sup.2+], and voltage independent, with an average channel activity ([NP.sub.o]) of 1.02 at command potential (-[V.sub.p]) ranging from 0 to -80 mV. In summary, MC exhibited an electrogenic [Ca.sup.2+] influx pathway that is suggestive of [Ca.sup.2+] entry through SOC, as well as a small-conductance divalent-selective channel displaying biophysical properties consistent with SOC. Based on estimates of whole cell [Ca.sup.2+] influx derived from our data, we conclude that SOC with low single-channel conductance must be highly abundant in MC to allow significant capacitative [Ca.sup.2+] entry in response to depletion of the intracellular store. cadmium; lanthanum; fura 2 fluorescence; patch clamp; thapsigargin; angiotensin II