Stimulation of liver heme oxygenase in hexachlorobenzene-induced hepatic porphyria.

We have measured liver heme oxygenase, a heat shock protein known to be increased under conditions of oxidative stress, to obtain additional evidence for an oxidative stress mechanism in hepatic uroporphyria induced by hexachlorobenzene (HCB). We have studied heme oxygenase at different times during HCB treatment and in two strains of rats (Agus and Wistar strains), which are known to differ in their sensitivity to the porphyria-inducing properties of HCB, in order to ascertain whether the same time course and genetic differences known to exist for the induction of porphyria also apply to hepatic oxidative stress. HCB induced heme oxygenase and accumulation of porphyrins in the liver of rats of both strains; no significant difference was found between the two strains in the HCB-induced heme oxygenase activity. The increased activity of the enzyme was first detected during the early phases of treatment, when a modest increase in liver porphyrins was observed; heme oxygenase remained at induced levels for several weeks during HCB treatment, and was still raised when an increase in total liver iron content and the onset of marked porphyria were also found. In contrast to the effects of HCB, phenobarbitone sodium (given in the drinking water for up to 4 weeks) produced similar elevations of total liver cytochrome P450 as HCB, but did not stimulate heme oxygenase or increase the total liver content of either iron or porphyrins. These results are compatible with an oxidative stress mechanism in HCB-induced liver toxicity and porphyria, but also suggest the existence of successive stages in the induction of hepatic porphyria, with more than one mechanism contributing to the marked accumulation of uroporphyrin.

Hepatic function in workers occupationally exposed to carbon tetrachloride.

OBJECTIVES: To identify any differences in hepatic function between workers exposed to carbon tetrachloride and controls, and to identify the best variable with which to examine any effects. METHODS: In a cross sectional study of hepatic function in workers occupationally exposed to carbon tetrachloride, 135 exposed employees were compared with 276 non-exposed controls. The exposed group was taken from three sites in the north west of England and the control group included non-exposed workers from one of these sites and another site located nearby. Demographic and alcohol consumption data were collected from both groups by questionnaire. Each member of the study group was allotted a notional estimated exposure to carbon tetrachloride, calculated from historic personal monitoring data and job category. A fasting sample of blood was taken from all participants and analysed for a variety of biochemical and haematological variables. The techniques of univariate and multivariate analysis of variance were used to investigate the effect on biochemical and haematological indices of a range of factors. RESULTS: Multivariate analysis of variance of four core liver function variables, alanine transaminase, aspartate transaminase, alkaline phosphatase, and gamma-glutamyl transferase, showed a significant difference between exposed and non-exposed workers. The univariate analyses identified increases in only alkaline phosphatase and gamma-glutamyl transferase within the exposed group and these did not show a significant dose-response relation. Univariate analysis of variance did show effects of alcohol and age on several variables. Significant differences between exposed and control groups for three haematological variables, haemoglobin, packed cell volume, and red blood count, were thought not to be due to the effects of exposure. Clinical review of exposed subjects with abnormal results did not show clinically evident disease that could have been associated with exposure to carbon tetrachloride. Also, a follow up study conducted three years after the cross sectional study at the site with highest exposures to carbon tetrachloride showed no evidence of any further changes in liver function variables. CONCLUSIONS: The most sensitive statistical methods have shown significant differences in the liver function variables measured between people exposed to carbon tetrachloride and the control group. The interpretation of the data collected was that these differences may be due to exposure to carbon tetrachloride but this was not clearly shown. Furthermore, the changes found have not given rise to any clinical disease.

Induction of trimethyltin neurotoxicity by dietary administration.

Groups of 12 male and 12 female rats were fed diets containing 0 or 8ppm trimethyltin chloride for up to 25 days. All the animals were observed prior to the study start and daily throughout the study for any changes in clinical condition. In addition, detailed clinical observations, including quantitative assessments of landing foot splay, sensory perception, muscle weakness and locomotor activity were monitored during the study. At the end of the study, the rats were killed and subjected to a full Post-mortem. Selected nervous system tissues were removed, processed and examined microscopically. Clinical signs typical of trimethyltin neurotoxicity (e.g. aggression, shaking and convulsions) were seen in rats receiving diets containing 8ppm trimethyltin chloride for as little as 22 days. Neuropathological lesions consisting of extensive neuronal cell necrosis in the limbic region of the brain, vacuolar degeneration of ventral horn cells of the spinal cord and a marginal increase in Wallerian-type degeneration were seen. The study demonstrates that trimethyltin neurotoxicity can be induced by dietary administration and that both male and female rats are equally sensitive.

Acute testicular toxicity of 1,3-dinitrobenzene and ethylene glycol monomethyl ether in the rat: evaluation of biochemical effect markers and hormonal responses.

The studies described in this paper were undertaken to evaluate the use of plasma enzymes of testicular origin and plasma hormones as markers of acute testicular toxicity. Rats were dosed by gavage with a single dose of either 1,3-dinitrobenzene (1,3-DNB) or ethylene glycol monomethyl ether (EGME). Two experimental designs were used: a dose response and a time-dose response course. Lactate dehydrogenase isozyme C4 (LDH-C4) and sorbitol dehydrogenase (SDH) were used as germ cell markers and leucine aminotransferase (LAT) and androgen binding protein (ABP) were used as Sertoli cell markers. Luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were also monitored. Histopathology confirmed the known testicular toxicity of 1,3-DNB and EGME. 1,3-DNB induced Sertoli cell damage with associated degenerative changes in late pachytene spermatocytes. The effects of EGME were mainly on early and late pachytene and dividing spermatocytes. No changes in either testicular or plasma SDH or LAT were found. Similarly no effects were observed for plasma LH or testosterone. However testicular LDH-C4 and testosterone, plasma LDH-C4, ABP, and FSH did show compound related effects. LDH-C4 was reduced in testis and increased in plasma with both compounds and plasma LDH-C4 remained elevated up to 14 days after dosing. ABP levels in plasma were increased with 1,3-DNB and EGME. A reduction in testicular testosterone levels was recorded and plasma FSH concentrations were elevated after EGME treatment. It is concluded that plasma LDH-C4 activity and ABP may be of diagnostic value in acute testicular toxicity. Increases in plasma LDH-C4 precede noticeable histological findings.

Assessment of renal function and damage in animal species. A review of the current approach of the academic, governmental and industrial institutions represented by the Animal Clinical Chemistry Association.

There are a wide variety of laboratory tests available to assess damage to and functional impairment of the kidneys, although the effectiveness of these tests varies greatly depending upon the site specificity of the damage and to a lesser extent upon the animal species involved. Several traditional tests of renal dysfunction and damage, including plasma creatinine and urea, and urinalysis (dipstick and/or quantitative protein), can be used in the first instance to detect nephrotoxicity. A second tier of specific, targetted indicators (concentration test, urinary enzymes, clearance of analytes, specific proteins, etc.) may then be applied to identify further the site of the lesion and the functional status of the kidneys. The glomerular filtration rate (GFR) may be estimated from the clearance of exogenous and endogenous substances. The difficulty in obtaining accurately timed urine samples limits the value of these tests in small animals, although methods that do not involve urine collection are available. The kidney is the origin of several enzymes found in urine that can be used to monitor the toxic effects of chemicals and therapeutic substances. Selective measurement of enzyme activities in urine can be used to detect the site of the renal lesion after traditional tests have established the presence of renal injury. Separation of proteins in urine by electrophoretic techniques may also be used to discriminate damage to different parts of the nephron. Renal cell excretion in urine is a sensitive but unreliable indicator of acute damage to the proximal tubule. The rate of cell excretion is not a good predictor of the severity of tubular injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Urinary enzymes and protein patterns as indicators of injury to different regions of the kidney.

Acute experimental models of renal damage to the proximal tubular, glomerular, and papillary regions of the rat were produced by administration of hexachloro-1:3-butadiene (HCBD), puromycin aminonucleoside (PAN), and 2-bromoethylamine (BEA), respectively. Several routine indicators of nephrotoxicity, the enzymes alkaline phosphatase and N-acetyl-beta-glucosaminidase, and the molecular weight of protein excretion were determined on urine samples. Tubular damage produced by HCBD or BEA was discriminated both quantitatively and qualitatively from glomerular damage produced by PAN. The latter was characterized by a pronounced increase in protein excretion, especially proteins with molecular weight greater than 40,000 Da. In contrast, protein excretion in tubular damage was raised only slightly and characterized by excretion of proteins of a wide range of molecular weights. Proximal tubular damage caused by HCBD and papillary damage caused by BEA were distinguished both by conventional urinalysis (volume and specific gravity) and by measurement of the two urinary enzymes. Alkaline phosphatase and glucose were markedly and transiently elevated in proximal tubular damage and N-acetyl-beta-glucosaminidase showed a sustained elevation in papillary damage. It is concluded that both selective urinary enzymes and the molecular weight pattern of urinary proteins can be used to provide diagnostic information about the possible site of renal damage.

The induction of omega and beta-oxidation of fatty acids and effect on alpha 2u globulin content in the liver and kidney of rats administered 2,2,4-trimethylpentane.

The effect of daily administration of 12 mmol/kg 2,2,4-trimethylpentane for 10 d on hepatic and renal microsomal mono-oxygenase activity, peroxisomal beta-oxidation and the concentration of alpha 2u-globulin has been examined in male and female rats. 2,2,4-Trimethylpentane produces liver and, to a lesser extent, kidney enlargement. This is associated with the selective induction of cytochrome P-450-mediated omega-oxidation and peroxisomal beta-oxidation of fatty acids and proliferation of peroxisomes. Male rats show a more marked response than female rats. 2,2,4-Trimethylpentane produces an increase in alpha 2u-globulin in the kidney of male rats. The relevance of selective induction of omega- and beta-oxidation of fatty acids and accumulation of alpha 2u-globulin to renal tubular necrosis in male rats requires further study.

Alpha 2U-globulin: measurement in rat kidney and relationship to hyaline droplets.

The concentration of renal alpha 2U-globulin increased in a dose-dependent manner in adult male but not female rats which received a single dose of 2,2,4-trimethylpentane (TMP). After administration of a single dose of 12 mmol TMP/kg to adult male rats, the renal concentration of alpha 2U-globulin reached a peak at 48 hours and returned to near background level after 7 days. These changes in renal alpha 2U-globulin concentration were closely paralleled by changes in renal hyaline droplet formation. Renal alpha 2U-globulin and hyaline droplets were absent in normal pre-puberty male rats, and neither could be stimulated by a single dose of TMP. alpha 2U-Globulin was localised in the renal cortex of adult male rats, in particular the S2 segment of the proximal tubule. A greater staining intensity due to alpha 2U-globulin was seen in the S2 and adjacent segments after a single dose of TMP. A strong association is suggested between the presence of renal hyaline droplets and the occurrence of alpha 2U-globulin.

Alpha 2U-globulin: measurement in rat kidney following administration of 2,2,4-trimethylpentane.

Hyaline droplet formation was stimulated markedly in the kidneys of post-puberty male rats 24-48 h after a single oral dose of 12/24 mmol/kg 2,2,4-trimethylpentane [TMP]. Renal hyaline droplet formation could not be detected in female rats or in pre-puberty male rats following similar doses of TMP. A dose-dependent increase in the renal concentration of the androgen-dependent low molecular weight protein, alpha 2U-globulin was observed in post-puberty male rats 24 h after a single oral dose of TMP, over the range 0.3-12.0 mmol/kg. After administration of a single dose of 12 mmol/kg TMP to male rats, the renal concentration of alpha 2U-globulin rose steadily up to a peak after 48 h and then returned slowly to near normal after 7 days. Renal alpha 2U-globulin could not be detected in female rats and in pre-puberty male rats. An immunocytochemical assay was developed to examine the distribution of alpha 2U-globulin within the kidney. alpha 2U-Globulin was localised primarily in the S2 segment of renal proximal tubules in untreated male rats. Rats which received a single dose of 12 mmol TMP/kg showed not only a greater staining intensity, due to the presence of a higher concentration of alpha 2U-globulin, but also staining in adjacent segments of the renal cortex. Several urinary biochemical indicators of nephrotoxicity were measured daily in male rats for up to 72 h following a single dose of 12 mmol TMP/kg. Renal proximal tubular function was unimpaired by TMP treatment. On the basis of studies in untreated and TMP-treated rats, a strong association has been found between the presence of renal hyaline droplets and the occurrence of renal alpha 2U-globulin. The findings in the present study provide an explanation for the occurrence of renal hyaline droplets only in adult male rats, but do not, as yet, establish the toxicological significance of increases in renal hyaline droplet formation.

Failure of inorganic lead exposure to impair natural killer (NK) cell and T lymphocyte function in rats.

Natural killer (NK)-cell and T-cell functions have been measured in rats following exposure to either 100 or 1000 ppm lead as lead acetate in drinking water. The capacity of splenocytes isolated from exposed animals to mediate native and interferon (IFN)-activated natural cytotoxicity was measured at three effector:target-cell ratios and found to be normal at all time points examined. Furthermore, splenic T-cell function of treated animals as determined by phytohaemagglutinin (PHA)-induced proliferation was comparable to control values. We conclude that low-level lead exposure is not associated with an impairment of either T-or NK-cell function.

Electrophoretic variants of alpha 2u-globulin in the livers of adult male rats: a possible polymorphism.

Two-dimensional polyacrylamide gel electrophoresis has been used to examine the microsomal fractions from the livers of 32 adult male Alpk/AP (Wistar-derived) rats for the presence of alpha 2u-globulin variants of differing isoelectric point. Three major such isoelectric variants are described. Different combinations of these three forms were found in the population examined, with one-half of the animals expressing all three variants in approximately equal proportions and with one variant being present in all the animals examined. An understanding of the relevance of such different alpha 2u-globulin profiles to the individual animal must now await the assignment of a biological role for alpha 2u-globulin.

Influence of chronic low-level exposure to lead on plasma immunoglobulin concentration and cellular immune function in man.

The immunological status of individuals occupationally exposed to low levels of inorganic lead has been examined and compared with that of non-exposed, age and sex-matched controls. At the time of testing the exposed population had a mean (+/- SD) blood lead concentration of 38.4 +/- 5.6 micrograms X 100 ml-1 (n = 39) compared with a mean value of 11.8 +/- 2.2 micrograms X 100 ml-1 (n = 21) for the control group. No differences in the serum concentrations of IgG, IgA and IgM between the populations were observed and there existed no correlation between blood lead concentration and serum immunoglobulin levels. In addition assessment was made of the capacity of peripheral blood mononuclear cells to respond to the mitogen phytohaemagglutinin (PHA), a correlate of T cell function, and to spontaneously lyse cells of the erythroleukaemic cell line K562, a measure of NK cell function. In neither case was there a difference between exposed and control populations and no correlation between reactivity and blood lead concentration. Although previous studies in rodents have indicated that exposure to inorganic lead resulting in similar blood lead concentrations may compromise immune competence our data suggest that no similar effect occurs in man.

The absence of testicular atrophy and in vivo and in vitro effects on hepatocyte morphology and peroxisomal enzyme activities in male rats following the administration of several alkanols.

Previous studies have shown that ethylhexanol (2-EH) and its oxidation products, but not n-hexanol, produce hepatomegaly, peroxisomal proliferation and hypotriglyceridaemia. In the present studies we have confirmed that at 1 mmol/kg doses, neither the linear nor branched chain alcohols induce testicular atrophy, hepatomegaly, peroxisome proliferation or hypolipidaemia. In vivo, neither the free alcohols nor their metabolic products seem to be responsible for the activity of the parent plasticiser. The released monoesters are probably the more potent metabolic products responsible for the hepatomegaly, peroxisomal proliferation and hypolipidaemia. This contention is supported by the in vitro hepatocyte data which demonstrate the induction of peroxisomal oxidative enzymes by MEHP whereas the alcohols were without effects.

The pathogenesis and effect on renal function of nephrocalcinosis induced by different diets in female rats.

The ability of three diets to produce nephrocalcinosis in female rats has been compared after 30, 60 and 90 days of feeding. Severe nephrocalcinosis with associated tubular damage was induced with a standard laboratory maintenance low-protein diet and with a diet containing a single-cell protein (Pruteen) but not with a casein-containing diet. Both the Pruteen and casein diets were high-protein diets. The development of the lesion was equally rapid following feeding of either the laboratory maintenance diet or the Pruteen diet. Nephrocalcinosis was present, with body kidneys similarly affected, at each 30-day interval with either diet. A severe degree of nephrocalcinosis was associated with widespread renal damage although signs of tubular regeneration and repair were evident at 30 days. Nephrocalcinosis induced by the Pruteen diet was accompanied by enlargement of the kidney. Renal function was assessed at each time by the measurement of urine volume, specific gravity and protein content, inulin clearance, and the excretion of the urinary enzymes N-acetyl-beta-glucosaminidase and alkaline phosphatase. Despite extensive calcification and some tubular damage, renal function was unimpaired, reflecting the capacity of the functional reserve of the kidney.

An evaluation of renal function in workers occupationally exposed to mercury vapour.

The renal function of a population of workers occupationally exposed to mercury in the chlor-alkali industry has been examined and compared to that of a population of workers with no occupational exposure to mercury. Measurement of specific urinary proteins and enzymes have been carried out on each individual on three separate occasions and have been complemented by blood plasma measurements at the final visit. Under the conditions of exposure to mercury sustained in this study, there is no evidence of an increased prevalence of renal dysfunction as indicated by enzyme and protein measurements. The urinary concentration of the low molecular weight protein, beta 2-microglobulin, is significantly lower in the mercury-exposed group than in the control group. In contrast to recently published literature, no relationship is seen between urinary mercury concentration and the appearance of high molecular weight protein in urine. A small increase in the prevalence of higher activities of the urinary enzyme N-acetyl-beta-glucosaminidase and gamma glutamyl transferase is observed when the urinary mercury concentration exceeds 100 micrograms/g creatinine. A small increase in the prevalence of raised urinary N-acetyl-beta-glucosaminidase activity is observed when the duration of exposure to mercury exceeds ten years. The pattern of proteinuria has been characterised in a total of sixteen individuals from both populations; a low molecular weight proteinuria is seen in three individuals from the control group whilst a high molecular weight proteinuria is seen in the remainder (seven in the control and six in the mercury group).

Influence of dietary cadmium on the distribution of the essential metals copper, zinc and iron in tissues of the rat.

The effect of long-term dietary cadmium treatment upon the distribution of the metals copper, iron and zinc has been compared in various organs of male and female rats. The renal accumulation of cadmium was similar in both sexes without a plateau being reached. In contrast, the hepatic accumulation of cadmium was higher in the female than in the male rat and a plateau was observed after 30-35 weeks of dietary cadmium treatment. Most of the cadmium which accumulated in these organs was recovered in the metallothionein fraction andthe concentration of hepatic cadmiumthionein in the female rat was correspondingly higher than in the male rat. Accumulation of cadmium was associated with an increased zinc concentration in the liver and an increased copper concentration in the kidney; these increases were correlated with increases in liver and kidney metallothioneins induced by cadmium. Uptake of cadmium into organs other than liver and kidney occurred to a small extent but was not associated with changes in the concentration of copper and zinc. Cadmium also accumulated in the intestinal mucosa where it could be recovered in a fraction corresponding to metallothionien. A loss of iron from the liver and kidney was also observed following dietary cadmium treatment and involved mainly a loss of iron from ferritin.

Different patterns of hepatic microsomal enzyme activity produced by administration of pure hexachlorobiphenyl isomers and hexachlorobenzene.

Three hexachlorobiphenyl isomers, 2,2',4,4',5,5'-hexachlorobiphenyl (I), 2,2',3,3',4,4'-hexachlorobiphenyl (II) and 2,2',3,4,4',5'-hexachlorobiphenyl (III), have been administered to rats and the effects of these three compounds upon hepatic microsomal drug metabolism and upon hepatic porphyrins have been studied. Comparisons have been made with hexachlorobenzen and a commercial polychlorinated biphenyl mixture, Aroclor 1254. From measurements of activities of microsomal drug oxidations in vitro, the durations of pharmacological actions of certain drugs in vivo and spectral shifts associated with cytochrome P-450 it is shown that the three pure hexachlorobiphenyl isomers initially produce changes in hepatic microsomal activity which resemble those seen after treatment with phenobarbitone (PB). In contrast, following chronic feeding of the isomers, compounds II and III but not I produce a pattern of hepatic microsomal enzyme activity which shows some characteristics of the 3-methylcholanthene (3-MC) and some characteristics of the phenobarbitone classes of inducer. Also, compounds II and III, but not I, cause accumulation in the liver of porphyrins containing either seven or eight carboxyl groups. These two responses are similar to those observed following hexachlorobenzene treatment and suggest that a relationship may exist between the mixed pattern of enzyme induction and the onset of hepatic porphyrin accumulation.

The effect of cobalt on the uptake of plasma iron by the liver.

1. After an intraperitoneal injection of 59Fe the recovery of radioactivity in the liver, but not in other tissues, was increased in cobalt-pretreated rats. There was no proportional increase in the radioactivity recovered from liver haem. 2. Rats were injected intravenously with serum containing protein-bound 59Fe and 125I-labelled albumin as a marker. At various times after injection the specific radioactivities of iron in plasma and of non-haem iron in liver were determined; corrections were applied for the content of plasma in samples of liver. In cobalt-pretreated rats there was a more rapid loss of 59Fe radioactivity from the plasma and a corresponding increase in the uptake of 59Fe into liver non-haem iron. 3. The results are discussed in relation to the possible sites of action of cobalt, and the possibility is considered that only a fraction of the liver non-haem iron may be involved.