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Archives of Toxicology (v.73, #7)
An iron-deficient diet stimulates the onset of the hepatitis due to hepatic copper deposition in the Long-Evans Cinnamon (LEC) rat by Naoki Sugawara; Chieko Sugawara (pp. 353-358).
To study effects of dietary Cu and Fe levels on the onset of hepatitis in Long-Evans Cinnamon (LEC) rats, female rats (40 days old) were fed a semipurified diet containing 0.1 or 10 mg Cu/kg and 1.5 or 150 mg Fe/kg in a 2 × 2 factorial arrangement for 35 days. At 75 days after birth, LEC rats (+Cu−Fe) fed a Cu-sufficient but Fe-deficient diet (Cu, 10 mg/kg; Fe, 1.5 mg/kg) showed jaundice, with lethargy, anorexia, and malaise. The biochemical variables relating to liver function were significantly increased compared to three other groups, a Cu- and Fe-deficient (−Cu−Fe) group, a Cu-deficient but Fe-sufficient (−Cu+Fe) group, and a Cu and Fe sufficient (+Cu+Fe) group. Furthermore, the +Cu−Fe rat liver showed massive necrosis with huge nuclei. The other three groups presented no biochemical and histological findings of hepatitis. Hepatic Cu and metallothionein concentrations were 289 ± 87 (mean ± SD) μg/g liver and 8.7 ± 1.8 mg/g liver, respectively, in the +Cu−Fe rats. However, in the +Cu+Fe group the values were 196 ± 28 μg Cu/g liver and 10.8 ± 1.0 mg/g liver. Hepatic Fe deposition was not influenced significantly by the dietary Cu level. The +Cu−Fe group with jaundice showed the highest free Cu concentration in the liver among the four groups, but the hepatic free Fe concentration was similar to those in the −Cu+Fe and +Cu+Fe groups. Our results indicate that an Fe-deficient diet enhances the deposition of hepatic Cu due to increased absorption of Cu from the gastrointestinal tract. This deposition stimulated the onset of hepatitis.
Keywords: Key words Long-Evans Cinnamon rat; Cu and Fe metabolism; Cu and Fe deficient diets; Hepatitis; Wilson disease
Aluminum toxicity perturbs long bone calcification in the embryonic chick by Conrad E. Firling; Theresa A. Hill; Arlen R. Severson (pp. 359-366).
Long bone calcification in chick embryos acutely- or chronically-treated with aluminum (Al) citrate was investigated. Acutely treated embryos received 100 μl of 60 mM Al citrate, 60 mM sodium (Na) citrate, or 0.7% sodium chloride on day 8 of incubation. Chronically treated embryos received a daily 25 μl dose of the above solutions beginning on day 8. Following 2–8 days of additional incubation, blood was collected, embryos killed, hind limbs radiographed, and tibias collected. Radiography indicated that Al administration resulted in a persistent angulation in the mid-diaphysis of tibias and femurs and a transient mineralization defect during the 10- to 12-day period of incubation. Tibias from 10- to 12-day embryos which were administered Al contained significantly less (P < 0.005) bone calcium (Ca) compared with tibias from NaCl-treated embryos. By day 14 there were no significant differences among the Ca content of tibias from embryos acutely treated with Al citrate, Na citrate or NaCl. Similarly, the rate of 45Ca uptake by tibias of embryos treated with Al was significantly lower on days 10 (acute) and 12 (chronic) with no significant differences in Ca uptake rate among the three treatment groups by day 16. In each treatment group bone alkaline phosphatase (ALPase) activity increased approximately tenfold between days 10 and 16. At all stages, bone ALPase activity was consistently higher and significantly different (chronic) compared with levels in NaCl-treated embryos. In contrast, Al had no significant effect on the rate of tibia collagen and noncollagenous protein synthesis or serum levels of procollagen carboxy-terminal propeptide (PICP), osteocalcin, and parathyroid hormone (PTH).
Keywords: Key words Aluminum toxicity; Embryonic bone; Bone calcification; Collagen; Alkaline phosphatase
Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones by Tadashi Furukawa; Sunao Manabe; Toshiyuki Watanabe; Shinya Sehata; Satoru Sharyo; Tadahiko Okada; Yuji Mori (pp. 367-372).
Hepatic P450 monooxygenase activities, which strongly influence the efficacy and/or toxicity of drugs, are known to fluctuate daily. We also know that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities fluctuate daily, with apparently high values during the dark period in male rats. However, there is little knowledge about the factors that regulate daily fluctuation of P450 monooxygenase activities. In the present study using rats, we induced lesions in the suprachiasmatic nucleus (SCN) of the brain, the known site of the body's internal clock, and examined the effects on the daily fluctuation of the ACD activities to clarify the relationship between the SCN and the daily fluctuation of P450 monooxygenase activities. In addition, adrenalectomy was performed to re-evaluate the influence of adrenal hormones on the P450 activities. Our results indicated that daily fluctuations of the hepatic ACD activities were completely eliminated in the SCN-lesioned rats. However, the ACD activities in the adrenalectomized rats showed apparent daily fluctuations with high values during the dark period and low values during the light period. Therefore, this study demonstrated that the daily fluctuation of the hepatic P450 monooxygenase activities in male rats is controlled by the SCN but remains unaffected by the adrenal hormones.
Keywords: Key words Alkoxycoumarin dealkylase; Liver; Daily fluctuation; Suprachiasmatic nucleus (SCN); Adrenal
The relationship between decrease in Cx32 and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis in the rat by Toshiyuki Shoda; Kunitoshi Mitsumori; Hiroshi Onodera; Kazuhiro Toyoda; Chikako Uneyama; Takayoshi Imazawa; Masao Hirose (pp. 373-380).
To examine the relationship between the decrease in connexin 32 (Cx32) and induction of P450 isozymes in the early phase of clofibrate hepatocarcinogenesis, a total of 20 male F344 rats were initiated with a single intraperitoneal injection of 150 mg/kg of diethylnitrosamine (DEN) or given the saline vehicle alone and starting 2 weeks later given diet containing 0.18, 0.09, and 0% clofibrate for 6 weeks. All animals were subjected to two-thirds partial hepatectomy at week 3 and killed at week 8. Absolute and relative (ratios to body weight) liver weights were significantly increased in the DEN + clofibrate groups compared with the DEN-alone group. Diffuse hepatocellular hypertrophy with granular cytoplasmic eosinophilia characterized by a marked increase in peroxisomes and smooth endoplasmic reticulum, was observed in the clofibrate treated rats. Induction of cytochrome P450 (CYP) 4A1 and 2B1/2 was noted in the DEN + clofibrate groups, this being most marked in the CYP 2B1 case. Immunohistochemically, positive immunostaining for anti-CYP 4A1 and CYP 2B1 were observed diffusely and centrilobularly, respectively. The numbers and areas of Cx32-positive spots per hepatocyte in the centrilobular areas in the treated rats were significantly decreased in an essentially dose-dependent manner, but no changes were observed in periportal areas. The numbers and areas of foci positive for glutathione S-transferase placental form (GST-P) were decreased in a dose dependent manner in the clofibrate treated groups. These results suggest that the CYP 2B1/2 induction and Cx32 decrease in centrilobular hepatocytes, similarly to those thought to be involved in the hepatic promotion mechanism of phenobarbital, may also play important roles in clofibrate actions in the liver, in addition to its causation of oxidative DNA injury.
Keywords: Key words Clofibrate; Connexin; Liver; Promotion; P450
Effect of dental materials on gluconeogenesis in rat kidney tubules by F. X. Reichl; J. Durner; H. Mückter; B. Elsenhans; W. Forth; K. H. Kunzelmann; R. Hickel; W. Spahl; W. R. Hume; G. W. Moes (pp. 381-386).
The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA) as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) on gluconeogenesis was investigated in isolated rat kidney tubules. From starved rats kidney tubules were prepared and isolated by digestion with collagenase. Every 10 min up to 60 min 1-ml samples were drawn from the cell suspension for quantitating the glucose content. Glucose formation in controls was 3.3 ± 0.2 nmol/mg · per min (mean ± SEM, n=21). Relative rates of glucose formation were obtained by expressing individual rates as a percentage of the corresponding control. X–Y concentration curves (effective concentration, EC) of the substances were calculated by fitting a four-parametric sigmoid function to the relative rates of glucose formation at various test concentrations. At the end of the incubation period cell viability was assessed by trypan blue exclusion. Cell viability decreased within the 60 min interval from 90 to approx. 80% (controls), <25 (HEMA), <20 (TEGDMA), <10 (MeHgCl), and <10% (HgCl2). Values of 50% effective concentration (EC50) were calculated from fitted curves. EC50 values were (mmol; mean ± SEM; n=4): HEMA, 17.7 ± 2.9; TEGDMA, 1.8 ± 0.2; MeHgCl, 0.018 ± 0.0005; and HgCl2, 0.0016 ± 0.0005. The toxic effect of HgCl2 was ∼1000 or 10 000 higher than that of the dental composite components TEGDMA or HEMA, respectively.
Keywords: Key words Triethyleneglycoldimethacrylate (TEGDMA); 2-Hydroxyethylmethacrylate (HEMA); Dental material; Gluconeogenesis; Kidney cells
Time dependence of chloroform-induced metabolic alterations in the liver and kidney of B6C3F1 mice by Sabrina Rossi; Simonetta Gemma; Laura Fabrizi; Emanuela Testai; Luciano Vittozzi (pp. 387-393).
The time course of some biochemical changes in the liver and in the kidney was studied in B6C3F1 male mice dosed with a single i.p. injection of 150 mg/kg body weight (b.w.) CHCl3. Hepatic and renal microsomal cytochrome P450 (P450) content and some related monooxygenase activities, CHCl3 oxidative and reductive metabolism, cytosolic reduced glutathione (GSH) content and serum markers of nephrotoxicity were measured. In the liver no biochemical changes were produced up to a week after chloroform treatment. On the contrary, the drug-metabolizing enzyme system in the kidney was dramatically and rapidly inactivated by chloroform treatment. Maximum loss of GSH (50%), P450 (80%) and of different enzymatic activities, including CHCl3 bioactivation, occurred during the first 5 h. These biochemical alterations are early effects, not secondary to morphological tissue changes. Kidney parameters, altered by chloroform treatment, returned to control values at different times: renal function markers became normal in 48 h; GSH levels were recovered at 96 h and the drug-metabolizing enzyme activities at longer times. The present results clearly show that repeated daily doses of chloroform, as those used in carcinogenicity tests, find renal tubular cells not at their physiological status, due to the changes produced by the first chloroform dose. Therefore the similarity in P450-dependent chloroform metabolism shown in vitro by hepatic and renal microsomes from untreated B6C3F1 male mice or in vivo in animals treated once, is lost during repeated treatments. These features should be considered in understanding the different susceptibility of the liver and the kidney to chloroform-induced tumours.
Keywords: Key words Chloroform toxicity; Metabolism; B6C3F1 mice; Liver; Kidney
Diethylnitrosamine exposure-responses for DNA ethylation, hepatocellular proliferation, and initiation of carcinogenesis in rat liver display non-linearities and thresholds by Gary M. Williams; Michael J. Iatropoulos; Alan M. Jeffrey; Feng-Qi Luo; Chung-Xiou Wang; Brian Pittman (pp. 394-402).
In previous exposure-response studies, we have documented non-linearities for some of the early effects in rat liver of diethylnitrosamine (DEN) and a near no-effect levels for initiation of promotable liver neoplasms at the lowest cumulative exposure of 0.5 mmol/kg body weight; this in spite of formation of DNA adducts and induction of hepatocellular altered foci (HAF). To extend these investigations, in an initiation segment, young male F344 rats were administered four exposures of DEN ranging from a cumulative total of 0.25 mmol, which is half of the previously used low exposure, up to 2 mmol per kg body weight, an effective initiating exposure. These exposures were achieved by once weekly intragastric instillations of one-tenth the total exposures for up to 10 weeks. The initiation segment was followed by a 4 week recovery segment, to allow for remission of acute and subchronic effects of DEN, after which the groups were maintained on 0.06% phenobarbital in the diet for 24 weeks to promote liver tumor development in order to assess initiation. During and after initiation and at the end of recovery, selected groups were studied for several crucial effects involved in hepatocarcinogenicity. The low exposure produced a low-level of DNA ethylation at both 5 and 10 weeks of exposure, measured as O4-ethylthymidine, the most persistent promutagenic ethylation product. At the 5 week interval, the adduct values of the higher exposures were less than proportional to the increment of exposure, suggestive of nonlinearity. Assessment of cellular proliferation by staining for proliferating cell nuclear antigen revealed that the lowest exposure did not increase the replicating fraction of hepatocytes during the initiation (10 weeks) or recovery (4 weeks) segments, whereas in the three higher exposure groups, proliferation was increased in relation to dose and time. Preneoplastic HAF expressing glutathione S-transferase-placental-type were present at low multiplicity in control livers and their multiplicity was increased in all exposure groups by the end of exposure, at which time the increase in the high exposure group was disproportionately greater than the increment of exposure. After phenobarbital administration in the promotion segment, all exposure groups exhibited further HAF increases at 39 weeks. At the end of the promotion segment, no hepatocellular neoplasm was found in 80 controls or in 40 rats in the low exposure group. In the mid-low exposure group, which was the previously studied low exposure, only one adenoma was found, yielding a 3% incidence, while in the two higher exposure groups, 32 and 80% of rats exhibited liver neoplasms, which were increased disproportionately greater than the increments of exposure. Thus, the findings document non-linearities of early DEN effects and at the lowest cumulative dose, a no-effect level (NEL) or threshold for initiation of promotable liver neoplasms. These findings provide a conceptual basis for understanding why low-level exposures to DNA-reactive carcinogens may convey no cancer risk.
Keywords: Key words Diethylnitrosamine; DNA damage; Cell proliferation; Hepatocellular altered foci; Liver neoplasms
Cytotoxicity and genotoxicity of capsaicin in human neuroblastoma cells SHSY-5Y by François Richeux; Marta Cascante; Rachid Ennamany; Dominique Saboureau; Edmond Ekué Creppy (pp. 403-409).
Capsaicin, a natural product of Capsicum species, induces excitation of pain perception at nociceptive terminals. Our previous studies have shown that capsaicin inhibits protein synthesis in cultured monkey kidneys cells (Vero cells) and in primoculture of rat astrocytes. We have now investigated the effect of capsaicin on human neuroblastoma cells SHSY-5Y. The cytotoxicity has been assessed by incorporation of [3H]L-leucine into cellular protein in the presence of capsaicin and the genotoxicity has been evaluated using the comet assay and the fragmentation assay after incubation of neuroblastoma cells with 25–100 μM capsaicin. The concentration required to inhibit 50% of the protein synthesis (IC50) was found to be 60 μM after incubation with the toxin during one cellular cycle (5 days) of SHSY-5Y. The results of the comet test and DNA fragmentation assay clearly suggest that capsaicin is able to induce DNA strand breaks already with concentrations in the range of 50 μM, corresponding to 29.3 μM of capsaicin not bound to alpha-1 acid glycoprotein. Several daily topical applications of preparations containing 0.075% of capsaicin could lead to blood capsaicin concentration of this order of magnitude following transdermal passage (5% of the total quantity applied). Because DNA strand breaks or DNA lesions may affect cellular functions, lead to cell death and/or mutagenesis, our data in case of inappropriate DNA repair may have important implications for the possible health threats of capsaicin, specially in the case of misuse of capsaicin preparations in pathological situations.
Keywords: Key words Capsaicin; Genotoxicity; Cytotoxicity; SHSY-5Y cells
Upregulation of heme oxygenase gene expression in rat lung epithelial cells following exposure to cadmium by Hiroki Kitajima; Seishiro Hirano; Kazuo T. Suzuki (pp. 410-412).
Inhalation exposure to cadmium (Cd) is associated with inflammatory lesion in the lung. In the present study we have investigated cytotoxic effects of Cd on immortalized rat lung epithelial cells (SV40-T2) and gene expression in those cells following in vitro exposure to sublethal concentrations of cadmium chloride (CdCl2). The polymerase chain reaction (PCR)-based subtraction method was used to find differentially expressed genes between control and Cd-exposed SV40-T2 cells. The most prominent cDNA on an agarose gel was identical to a fragment of rat heme oxygenase (HO) gene. Northern blot analysis indicated that the level of HO mRNA expression in SV40-T2 cells was increased to 38-fold of the control value following exposure to 2.5 μM CdCl2 for 4 h. These results suggest that HO gene expression is one of the most sensitive biomarkers for acute exposure to Cd in the lung.
Keywords: Key words Cadmium; Lung epithelial cells; Polymerase chain reaction (PCR)-based subtraction; Heme oxygenase; Northern blot