Mary E. Davis

Effects of AT-125 on the nephrotoxicity of hexachloro-1,3-butadiene in rats

The role of gamma-glutamyl transpeptidase (gamma-GTP) in the nephrotoxicity of hexachloro-1,3-butadiene (HCBD) was studied using male Sprague-Dawley rats pretreated with AT-125 (Acivicin; L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid). Inhibition of gamma-GTP by more than 95% did not affect urine output, glomerular filtration rate, or tubular reabsorption of filtrate, sodium, or glucose. Nephrotoxicity observed during the first 24 hr after HCBD was not decreased by inhibition of gamma-GTP and beyond 24 hr nephrotoxicity was increased, rather than decreased, in the AT-125-pretreated group. HCBD impairs glucose reabsorption and this was greatly increased in the AT-125-pretreated group, indicating that function of the initial segment of the nephron is impaired by HCBD. Since inhibition of gamma-GTP did not protect against HCBD nephrotoxicity, it is concluded that gamma-GTP inhibition does not limit the formation of metabolites(s) which cause HCBD nephrotoxicity. Therefore, distribution of gamma-glutamyltranspeptidase does not account for the selective nephrotoxicity of hexachloro-1,3-butadiene.

Multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of Cr(VI)-transformed lung cells.

B-cell lymphoma-2 (Bcl-2) is an antiapoptotic protein known to be important in the regulation of apoptosis in various cell types. However, its role in malignant transformation and tumorigenesis of human lung cells is not well understood. We previously reported that chronic exposure of human lung epithelial cells to the carcinogenic hexavalent chromium Cr(VI) caused malignant transformation and Bcl-2 upregulation; however, the role of Bcl-2 in the transformation is unclear. Using a gene silencing approach, we showed that Bcl-2 plays an important role in the malignant properties of Cr(VI)-transformed cells. Downregulation of Bcl-2 inhibited the invasive and proliferative properties of the cells as well as their colony forming and angiogenic activities, which are upregulated in the transformed cells as compared to control cells. Furthermore, animal studies showed the inhibitory effect of Bcl-2 knockdown on the tumorigenesis of Cr(VI)-transformed cells. The role of Bcl-2 in malignant transformation and tumorigenesis was confirmed by gene silencing experiments using human lung carcinoma NCI-H460 cells. These cells exhibited aggressive malignant phenotypes similar to those of Cr(VI)-transformed cells. Knockdown of Bcl-2 in the H460 cells inhibited malignant and tumorigenic properties of the cells, indicating the general role of Bcl-2 in human lung tumorigenesis. Ingenuity Pathways Analysis (IPA) revealed potential effectors of Bcl-2 in tumorigenesis regulation. Additionally, using IPA together with ectopic expression of p53, we show p53 as an upstream regulator of Bcl-2 in Cr(VI)-transformed cells. Together, our results indicate the novel and multifunctional role of Bcl-2 in malignant transformation and tumorigenesis of human lung epithelial cells chronically exposed to Cr(VI).

Subacute toxicity of trichloroacetic acid in male and female rats

Trichloroacetic acid, TCA, is a water chlorination by-product similar to dichloroacetic acid, DCA. Because DCA has been shown to have effects on intermediary metabolism, TCA was tested to determine if it possesses similar capabilities. The effects were more pronounced in females. High doses of TCA (2.45 mumol/kg three times) decreased plasma glucose and lactate concentrations and liver lactate concentration. DCA had similar, less pronounced effects. In males DCA and TCA each decreased plasma lactate concentrations. Rats were exposed to TCA in drinking water for 14 days. The highest concentration (2.38 g/l) caused decreases of water and food consumption and loss of body weight. At 7 days females had decreased urine volume accompanied by a modest increase of urine osmolality, resulting in a significant decrease of excretion of solute. Concentrations of glucose in plasma and lactate in tissues were not significantly affected by this subchronic TCA exposure. These results indicate that TCA may have effects on intermediary metabolism similar to those of DCA.

Dichloroacetic acid and trichloroacetic acid increase chloroform toxicity.

Dichloro- and trichloroacetic acids (DCA and TCA) and chloroform are formed during chlorination disinfection of drinking water. The effects of DCA and TCA treatment on CHCl3 toxicity were assessed in these studies. Male and female rats were gavaged with DCA or TCA (0.92 and 2.45 mmol/kg administered 3 times over 24 h). Three hours after the last dose CHCl3 was injected ip (0.75 mg/kg). Male rats experienced some weight loss (15%) and slight increases of ALT and BUN, but there were no effects of either DCA or TCA on any of these responses. In females, CHCl3 increased plasma ALT and this response was greater (up to threefold) in the DCA group, compared to saline controls. Similarly, BUN was increased by CHCl3 and this was more severe (up to threefold) in both the DCA and TCA pretreated groups. These results show that CHCl3 toxicity is increased by DCA and TCA, and this effect is gender-specific, occurring only in females. DCA increases both liver and kidney toxicity, whereas TCA affects only kidney toxicity.

Delineation of in vitro chondrogenesis of human synovial stem cells following preconditioning using decellularized matrix

As a tissue-specific stem cell for chondrogenesis, synovium-derived stem cells (SDSCs) are a promising cell source for cartilage repair. However, a small biopsy can only provide a limited number of cells. Cell senescence from both in vitro expansion and donor age presents a big challenge for stem cell based cartilage regeneration. Here we found that expansion on decellularized extracellular matrix (dECM) full of three-dimensional nanostructured fibers provided SDSCs with unique surface profiles, low elasticity but large volume as well as a fibroblast-like shape. dECM expanded SDSCs yielded larger pellets with intensive staining of type II collagen and sulfated glycosaminoglycans compared to those grown on plastic flasks while SDSCs grown in ECM yielded 28-day pellets with minimal matrix as evidenced by pellet size and chondrogenic marker staining, which was confirmed by both biochemical data and real-time PCR data. Our results also found lower levels of inflammatory genes in dECM expanded SDSCs that might be responsible for enhanced chondrogenic differentiation. Despite an increase in type X collagen in chondrogenically induced cells, dECM expanded cells had significantly lower potential for endochondral bone formation. Wnt and MAPK signals were actively involved in both expansion and chondrogenic induction of dECM expanded cells. Since young and healthy people can be potential donors for this matrix expansion system and decellularization can minimize immune concerns, human SDSCs expanded on this future commercially available dECM could be a potential cell source for autologous cartilage repair.

Mechanism of allyl formate-induced hepatotoxicity in rainbow trout

Hepatotoxicity of allyl formate (AF) was studied in trout, to characterize the response of the teleost liver to a mammalian periportal hepatotoxicant. A dose-dependent decrease in liver nonprotein sulfhydryl (NPSH) concentration was observed at 3, 6, and 24 hr following 9.5, 28, and 95 mg/kg) AF with maximal depression seen at 6 hr (51, 40, and 29% control, respectively). Further evidence for glutathione (GSH) protection against AF toxicity was seen when diethylmaleate, a GSH depleting agent (0.6 ml/kg ip), administered 30 min prior to AF (9.5 and 28 mg/kg), increased AF hepatotoxicity (10-fold shift in the dose-response effect on SGPT). Also, N-acetyl-L-cysteine (150 mg/kg ip), a GSH precursor, protected liver against AF toxicity when injected 5 min prior to and 1, 5, and 9 hr after AF (28 and 95 mg/kg). Pyrazole (375 mg/kg ip), an alcohol dehydrogenase inhibitor, given 4 hr before AF (95 mg/kg), attenuated the histopathological effect of AF. These results indicate that AF, once bioactivated by alcohol dehydrogenase, causes significant toxicity in trout liver. GSH protects against AF-induced effects since greater than 50% decreases in liver GSH are required before toxicity is expressed.

A1 adenosine receptor deficiency or inhibition reduces atherosclerotic lesions in apolipoprotein E deficient mice

AIMS The goal of this study was to determine whether the A1 adenosine receptor (AR) plays a role in atherosclerosis development and to explore its potential mechanisms. METHODS AND RESULTS Double knockout (DKO) mice, deficient in the genes encoding A1 AR and apolipoprotein E (apoE), demonstrated reduced atherosclerotic lesions in aortic arch (en face), aortic root, and innominate arteries when compared with apoE-deficient mice (APOE-KO) of the same age. Treating APOE-KO with an A1 AR antagonist (DPCPX) also led to a concentration-dependent reduction in lesions. The total plasma cholesterol and triglyceride levels were not different between DKO and APOE-KO; however, higher triglyceride was observed in DKO fed a high-fat diet. DKO also had higher body weights than APOE-KO. Plasma cytokine concentrations (IL-5, IL-6, and IL-13) were significantly lower in DKO. Proliferating cell nuclear antigen expression was also significantly reduced in the aorta from DKO. Despite smaller lesions in DKO, the composition of the innominate artery lesion and cholesterol loading and efflux from bone marrow-derived macrophages of DKO were not different from APOE-KO. CONCLUSION The A1 AR may play a role in the development of atherosclerosis, possibly due to its pro-inflammatory and mitogenic properties.

Changes of hexachlorobutadiene nephrotoxicity after piperonyl butoxide treatment

The effects of piperonyl butoxide on hexachlorobutadiene (HCBD) nephrotoxicity were measured. The time course and severity of toxicity were affected. Five hours after either piperonyl butoxide or HCBD glomerular filtration rate (GFR) was decreased; at 24 h GFR had recovered for the piperonyl butoxide group but continued to fall in the HCBD group. The group treated with piperonyl butoxide and HCBD had the same GFR as the group treated with just HCBD. At 24 h after HCBD the piperonyl butoxide pretreated group was not different from the oil pretreated controls. At 48 h after HCBD, reabsorbtion of water and glucose was more severely impaired in the group pretreated with piperonyl butoxide. These results support the hypothesis that HCBD metabolites are involved in renal tubular dysfunction.

Alterations in the renal function of male and female rats exposed to maleic acid, dichloromaleic acid, and both compounds

Maleic acid (MA), a known nephrotoxicant in experimental animals, and its chlorinated derivative dichloromaleic acid (DCMA) are present in urban drinking water supplies as by-products of the chlorination process. This study was designed to characterize the effects of simultaneous exposure of subtoxic doses of DCMA and MA on renal function in both sexes of the Sprague-Dawley rat. Urine was collected at 24-h intervals from rats housed individually in stainless steel metabolism cages. Subcutaneous administration of MA at a dose of 150 mg/kg had no effect on several parameters of renal function in either sex at 24 h and only modest effects at 48 h. Renal slice studies showed that treatment of both male and female rats with DCMA (300 mg/kg) reduced p-aminohippurate (PAH) accumulation at 24 h with no effect on the uptake of tetraethylammonium ion (TEA). The combination of MA + DCMA caused a depression of TEA accumulation by slices from the female. Also, changes in urinary glucose excretion and blood urea nitrogen, although additive in the male following coexposure, appeared synergistic or potentiated in the female. These results suggest an enhanced susceptibility of the female rate to the nephrotoxic action of combined exposure to MA and DCMA.

Dichloroacetic acid pretreatment of male and female rats increases chloroform-induced hepatotoxicity

Dichloroacetic acid (DCA) and chloroform (CHCl3) are both major by-products of drinking water chlorination and DCA increases the hepatotoxicity of CHCl3. In this study, we further characterized this effect and investigated DCA-induced alterations of CHCl3 disposition and metabolism as a possible mechanism for this interaction. Both adult male and female Sprague-Dawley rats were gavaged with three doses (09:00, 16:00 and 09:00 the next morning) of DCA (each 2.45 mmol/kg), then challenged with an i.p. injection of CHCl3 (3.12 or 9.35 mmol/kg). Hepatic damage was assessed 24 h after CHCl3 administration as increased alanine aminotransferase (ALT), ornithine carbomyl transferase (OCT) and bilirubin in plasma. In a separate experiment, rats were pretreated with DCA or were given 14CHCl3 at the same dosages. The disposition of 14C in various tissues and covalent binding of 14CHCl3-derivatives to liver proteins and lipids were determined 1 h later. CHCl3-induced hepatotoxicity was significantly more severe in DCA-pretreated groups. ALT and OCT were more markedly elevated in DCA + CHCl3 (3.12 mmol/kg) groups than NaCl +CHCl3 animals. Plasma bilirubin content was elevated only in DCA + CHCl3 groups and females were more susceptible to this effect. The responses of rats to DCA treatment were somewhat gender-different. DCA treatment increased total cytochrome P450 in females, but not in males. Hepatic glutathione concentration was elevated in males after DCA treatment, but not in females. In the present study we confirmed that DCA pretreatment potentiates the CHCl3-hepatotoxicity of both male and female rats. However, there was little evidence that DCA pretreatment significantly affected CHCl3 disposition or increased CHCl3 binding in vivo.