Towards a unified and interdisciplinary model of ageing.

Researchers currently disagree about the appropriate biomarkers to monitor when measuring the ageing process. The major problem is identifying symptoms that are an end in and of themselves, from symptoms that are tied directly to the root cause, or causes, of ageing. This is most likely the reason that numerous, diverse and plausible theories for ageing co-exist. When young and old nuclei are exchanged between cells, the age of the resulting cell correlated with the nucleus. This suggests a large role of the nucleus as the target of ageing, although the sources of ageing may originate externally. There are three processes that occur when eukaryotes age. They are: (1) a progressive and patterned alteration of the structure of chromosomes after young adulthood has been reached, (2) a progressive and patterned malfunction of the degradation systems, and (3) age-altered post-translational modifications of proteins. A change in any one of these processes often causes a ripple effect that affects the other two processes. This paper begins by stating that the above three processes are the appropriate biomarkers of ageing. These three processes are coordinated with one another under normal physiological conditions. For example, proteasomes and their subunits have been found to regulate excision repair, transcription, and the turnover of nuclear/cytoplasmic receptors. The degradation system is also responsible for the removal of oxidized histones and other factors, which influence chromosome structure. Regulatory post-translational modifications at the histone level include methylation, phosphorylation, and acetylation. In addition, the above three processes undergo age related changes. Some of these modifications represent valid responses by the cell, but many do not. The effect of these age-altered macromolecules is perverse and unpredictable. For example, the cell's age-compromised degradation allows the accumulation of signaling complexes, which no longer match the needs of the cell. Age related histone and non-histone post-translational modifications alter both chromosome structure and expression. Nuclear pores have been found to slowly decrease in number in an age dependent manner. These pores have been found associated with the nuclear lamin. Several types of mutations in the lamin A gene cause progeria like symptoms. There is a diverse set of mechanisms that cause age related post-translational modifications. Previous attempts to find a commonality among those modifications have been disappointing. This paper will present a possible explanation that involves conformational changes caused by ionic and other perturbations in the nucleoplasm.

Urinary lead isotopes during pregnancy and postpartum indicate no preferential partitioning of endogenous lead into plasma.

We have compared lead isotopic ratios and lead concentrations in 51 matched blood and spot urine samples from 13 subjects covering the interval from before pregnancy through 180 days postpartum to evaluate whether mobilization of lead from the maternal skeleton is preferentially partitioned into plasma; we have used urine as an isotopic proxy for plasma. There was no statistically significant difference in the lead 206/lead 204 and lead 207/lead 206 ratios over pregnancy. The urine data for the postpartum period are in the opposite relationships to that predicted for a preferential partitioning hypothesis. These data provide no support for the hypothesis that lead released from the skeleton is preferentially partitioned into plasma.

Impact of diet on lead in blood and urine in female adults and relevance to mobilization of lead from bone stores.

We measured high precision lead isotope ratios and lead concentrations in blood, urine, and environmental samples to assess the significance of diet as a contributing factor to blood and urine lead levels in a cohort of 23 migrant women and 5 Australian-born women. We evaluated possible correlations between levels of dietary lead intake and changes observed in blood and urine lead levels and isotopic composition during pregnancy and postpartum. Mean blood lead concentrations for both groups were approximately 3 microg/dl. The concentration of lead in the diet was 5.8 +/- 3 microg Pb/kg [geometric mean (GM) 5.2] and mean daily dietary intake was 8.5 microg/kg/day (GM 7.4), with a range of 2-39 microg/kg/day. Analysis of 6-day duplicate dietary samples for individual subjects commonly showed major spikes in lead concentration and isotopic composition that were not reflected by associated changes in either blood lead concentration or isotopic composition. Changes in blood lead levels and isotopic composition observed during and after pregnancy could not be solely explained by dietary lead. These data are consistent with earlier conclusions that, in cases where levels of environmental lead exposure and dietary lead intake are low, skeletal contribution is the dominant contributor to blood lead, especially during pregnancy and postpartum.

Comparison of the rates of exchange of lead in the blood of newly born infants and their mothers with lead from their current environment.

Newly born infants (n = 15) were monitored for 6 months after birth or for longer periods to evaluate the changes in isotopic composition and lead concentration in infants as compared with that in women from the same population groups and to determine the clearance rates of lead from blood in the infants. These data represent the first published results for serial blood sampling in a relatively large cohort of newly born infants. Blood lead concentrations decrease from the cord to samples taken at 60 to 90 days and then increase by amounts varying from negligible to 166%. In spite of concern about individual susceptibility to lead pharmacokinetics, changes in isotopic ratio followed a smooth decrease over time for 9 of the 11 infants born to migrant parents, and the patterns of variation were quite reproducible. Data for 2 of 4 infants born to multigenerational Australian parents exhibited little change in isotopic ratio over time, and in the other two cases, the changes were attributed to diet. The rate of exchange (t1/2) for the migrant infants of lead in blood derived from the mother during pregnancy and the lead from the current environment was calculated by using a linear function and ranged from 65 to 131 (91+/-19, mean+/-SD) days. The half-lives for the exchange of skeletal and environmental lead for 7 of the 8 women before significant mobilization of lead from the maternal skeleton ranged from 50 to 66 (59+/-6) days. One explanation for the longer half-lives for infants as compared with the mothers may be the proportionally higher contribution of current environmental (Australian) lead in the infants at parturition. Exchanges of lead in infants are more complex than for the adults, reflecting inputs from sources such as maternal skeletal lead during breast feeding.

Relationships of lead in breast milk to lead in blood, urine, and diet of the infant and mother.

We have obtained stable lead isotope and lead concentration data from a longitudinal study of mobilization of lead from the maternal skeleton during pregnancy and lactation and in which the newly born infants were monitored for 6 months postpartum to evaluate the effects of the local environment on lead body burden of the infant. Samples of maternal and infant blood, urine, and diet and especially breast milk were measured for 21 mothers and 24 infants. Blood lead concentrations were less than 5 microg/dl in all except one subject. The mean lead concentration in breast milk +/- standard deviation was 0.73 +/- 0.70 microg/kg. In seven subjects for whom serial breast milk sampling was possible, the lead concentration varied by factors of from 2 to 4, and for three subjects there was an increase at or after 90 days postpartum. For the first 60-90 days postpartum, the contribution from breast milk to blood lead in the infants varied from 36 to 80%. Multiple linear regression analyses indicated statistically significant relationships for some of the variables of isotope ratios and lead concentrations between breast milk, blood, urine, and diet for infants and mothers. For example, the analyses revealed that both a mother's breast milk 207Pb/206Pb and 206Pb/204Pb ratios and lead concentration provide information to predict her infant's blood 207Pb/206Pb and 206Pb/204Pb ratios. The major sources of lead in breast milk are from the maternal bone and diet. An evaluation of breast milk lead concentrations published over the last 15 years indicates that studies in which the ratio of lead concentrations in breast milk to lead concentrations in whole maternal blood (Multiple>100) were greater than 15 should be viewed with caution because of potential contamination during sampling and/or laboratory analyses. Selected studies also appear to show a linear relationship between breast milk and maternal whole blood, with the percentage of lead in breast milk compared with whole blood of <3% in subjects with blood lead levels ranging from 2 to 34 microgram/dl. The levels of lead in breast milk are thus similar to those in plasma. Breast-fed infants are only at risk if the mother is exposed to high concentrations of contaminants either from endogenous sources such as the skeleton or exogenous sources.

Blood lead-urine lead relationships in adults and children.

To determine the potential for using instead of blood as an indicator of lead exposure, especially in infants, lead concentrations and high-precision lead isotopic measurements have been compared in venous blood and "spot" urine (n > 260 from 182 different subjects) collected within the same 24-h period. Physiological conditions for the children and most of the adults were considered to be in a steady-state between body stores and lead in the environment. In the case of some adults, conditions were initially not steady-state because exposure conditions changed (for example, subjects moved to a country with lead of different isotopic composition.) There was a high correlation (r2 = ) between the blood and urine measurements of the isotope ratios but about 10% of measurements were outliers--the blood and urine measurements were further apart than was consistent with the measurement error that was generally obtained. The discrepancy was usually found to be associated with the urine measurement and was attributed to contamination during sampling. Weekly urine and monthly blood monitoring of an adult male over a 24-month period showed and excellent correlations, although the standard deviations were about an order of magnitude higher than the precision measured for replicate analyses of a single blood or urine sample. "Spot" urine analyses for two male subjects gave excellent agreement with 24-h urine samples. Standard deviations of the spot analyses were of similar order to those in the 24-month monitored subject. In cases where female adults from Eastern Europe migrated to Australia, there was generally a more rapid exchange of skeletal lead with Australian environmental lead in urine compared with blood. These data do not support a differential partitioning of endogenous lead into the plasma. At this stage, isotopic measurements of urine can be used as a proxy for isotopic measurements in blood. However, lead concentrations in blood and in urine are only weakly related. Concentrations of lead in urine cannot serve to predict concentrations of lead in blood, particularly at the lower range of exposures, for example, at blood concentrations less than 10 microgram/d1.

Mobilization of lead from the skeleton during the postnatal period is larger than during pregnancy.

A cohort of 15 immigrant females to Australia and 7 native Australian controls were monitored on a monthly basis with high-precision lead isotopic methods during gestation and for 6 months after pregnancy to determine the extent of lead mobilization from the maternal skeleton. Quarterly environmental samples of house dust, drinking water, urban air, gasoline, and a 6-day duplicate diet were also measured. The geometric mean blood lead concentration for the immigrant females on arrival in Australia was 3.0 microg/dl (range: 1.9 to 20 microg/dl), and for the Australian controls was 3.1 gm/dl (range: 1.9 to 4.3 microg/dl). During gestation and after pregnancy, blood lead concentrations varied, with mean individual changes of -14% to 83%. For the immigrant subjects, the percentage change in blood lead concentration was significantly greater during the postpregnancy period than during the 2nd and 3rd trimesters (p < 0.001). Skeletal contribution to blood lead, based on the isotopic composition for the immigrant subjects, increased in an approximately linear manner during pregnancy. The mean increases for each individual during pregnancy varied from 26% to 99%. Skeletal lead contribution to blood lead was significantly greater (p < 0.001) during the postpregnancy period than during the 2nd and 3rd trimesters. The contribution of skeletal lead to blood lead during the postpregnancy period remained essentially constant at the increased level of lead mobilization, although the duration of breastfeeding varied from 1 week to more than 6 months. The increased contribution of skeletal lead to blood lead during the postpregnancy period is attributed to increased mobilization of lead from maternal skeletal stores during lactation. The increased contribution of skeletal lead both during pregnancy and in the postpregnancy period is consistent with increased bone resorption, and may be associated with an inadequate calcium intake observed in quarterly 6-day duplicate diets. Mobilization of skeletal lead stores represents a potentially important source of perinatal lead intake and accumulation in the developing fetus. Only two subjects consumed dietary supplements for calcium, and their mobilization of lead from the skeleton to the blood was the lowest of all the subjects. These two subjects' use of calcium supplements may have reduced mobilization of skeletal mineral stores to supply the calcium needs of pregnancy and lactation. Calcium supplementation may be an important means of limiting fetal exposure to lead.

Stable lead isotopes in teeth as indicators of past domicile--a potential new tool in forensic science?

A pilot study using stable lead isotope analyses has shown that permanent and deciduous teeth from Eastern and Southern European subjects have completely different lead isotopic compositions to those of Australian subjects. There are statistically significant differences between groups of teeth from subjects from the former Soviet Union (CIS), the former Yugoslavia, United Kingdom, and Lebanon. The isotopic analyses confirm the stability of lead in enamel but suggest that there is exchange of European lead with Australian lead in dentine amounting to about 1% per year. The isotopic differences in, and exchange of, European lead and Australian lead offer an exciting and powerful tool for forensic identification.

Pregnancy increases mobilization of lead from maternal skeleton.

The question of the extent of lead mobilization from the maternal skeleton during pregnancy and lactation is one of the most outstanding problems of lead toxicity. We have undertaken a longitudinal cohort study in an urban environment of European female immigrants of child-bearing age (18 to 35 years) to Australia whose skeletal lead isotopic composition has been determined to be different from that in their current environment. The cohort was to consist of 100 immigrants anticipated to provide 20 pregnant subjects who would be compared with two groups of control subjects: a matched immigrant nonpregnant control group and second-generation Australian pregnant control subjects. Pregnant subjects also serve as their own controls for a comparison of changes during gestation with those before conception. High-precision lead isotopic compositions and lead concentrations are measured in maternal blood and urine prenatally, monthly during gestation, and postnatally for 6 months; they are also measured in infant blood and urine for 6 months; environmental measures are sampled quarterly for 6-day duplicate diet, house dust and water, and urban air and gasoline. Because of continuing public health concerns about lead exposure, interim findings from this cohort are being reported. To date there have been 13 conceptions in immigrant subjects, with 7 births, in addition to 3 conceptions in the Australian control group, with 2 births. PbBs have been generally low, with a geometric mean of 3.0 microg/dl, and have ranged from 1.9 to 20 microg/dl. Increases in PbB of approximately 20% during pregnancy have been detectable even in subjects with low blood lead levels. The skeletal contribution to blood lead level, based on isotopic measurements, has exhibited a mean increase (and standard deviation) of 31% +/- 19% with a range from 9% to 65%. Earlier studies that used lead concentrations only have suggested that blood lead levels increased only during the second half of pregnancy. This increase in blood lead levels has also been observed in the present study. However, in two subjects the increases in total blood lead were also detected in the first 2 months of pregnancy. Changes in isotopic composition and blood lead during gestation for Australian pregnant controls were negligible. The ratio of cord/maternal blood lead levels varied from 0.54 to 1.05, and the ratio for the isotopic composition was 0.993 to 1.002. Results of this study confirm that lead is mobilized from skeletal stores at an accelerated rate during pregnancy and is transferred to the fetus. These results also show that mobilization from long-term stores (i.e., bone) contributes significantly to blood lead levels during pregnancy. Furthermore, exposure of the fetus to lead during pregnancy has implications for interpretations of neurobehavioral disorders attributed to only postnatal exposure. Even after 800 days of residence in Australia, the contribution of European skeletal lead to blood lead in nonpregnant subjects can be on the order of 50%, but the current PbB may give no indication of the former high skeletal lead burden.

Dietary lead intakes for mother/child pairs and relevance to pharmacokinetic models.

Blood and environmental samples, including a quarterly 6-day duplicate diet, for nine mother/child pairs from Eastern Europe have been monitored for 12 to >24 months with high precision stable lead isotope analysis to evaluate the changes that occur when the subjects moved from one environment (Eastern Europe) to another with different stable lead isotopes (Australia). The children were between 6 and 11 years of age and the mothers were between 29 and 37 years of age. These data were compared with an Australian control mother/child pair, aged 31 and 6 years, respectively. A rationale for undertaking this study of mother/child pairs was to evaluate if there were differences in the patterns and clearance rates of lead from blood in children compared with their mothers. Blood lead concentrations ranged from 2.1 to 3.9 microg/dl in the children and between 1.8 and 4.5 microg/dl in the mothers, but the mean of differences between each mother and her child did not differ significantly from zero. Duplicate diets contained from 2.4 to 31.8 microg Pb/kg diet; the mean+/- standard deviation was 5.5 +/- 2.1 microg Pb/kg and total daily dietary intakes ranged from 1.6 to 21.3 microg/day. Mean daily dietary intakes relative to body weight showed that the intake for children was approximately double that for the mothers (0.218 vs. 0. 113 microg Pb/kg body weight/day). The correlations between blood lead concentration and mean daily dietary intake either relative to body weight or total dietary intake did not reach statistical significance (p>0.05). Estimation of the lead coming from skeletal (endogenous) sources relative to the contribution from environmental (exogenous) sources ranges from 8 to 70% for the mothers and 12 to 66% for the children. The difference between mothers and children is not statistically significant (p = 0.28). The children do not appear to achieve the Australian lead isotopic profile at a faster rate than their mothers. These data provide evidence that the absorption or uptake of lead from dietary sources is similar in adult females and children of the age in this study. In spite of lower bone lead and faster bone remodeling and recycling in children compared with adult females, we see no differences between the mothers and their children in overall contribution of tissue lead to blood lead. Results from this study suggest that fractional absorption of ingested lead by children 6-11 years of age is comparable with absorption patterns observed among adult females in the 29-37-year-old age range. Because pharmacokinetic models apply a 40-50% absorption even for 7-year-old children, further investigations on fractional absorption of ingested lead by young children are warranted. Further investigations are especially needed in younger children than those who were subjects in the current study, particularly children in the 1-3-year-old age range. In addition, the effect of nutritional status and patterns of food intake on children's lead absorption require investigation, particularly given the increased prevalence of marginal nutritional status among low-income populations that are at increased risk of elevated blood lead levels.

Analysis of feed blends containing microencapsulated 2-ethyl-1-hexanol: verification of homogeneity and stability.

2-Ethyl-1-hexanol (2-EH) was nominated for carcinogenicity testing by the National Toxicology Program because it is a high-volume chemical and a major metabolite of di(2-ethylhexyl)phthalate, a known hepatocarcinogen and a known contaminant in blood storage bags. In addition to uses as an intermediate in the manufacture of plasticizers. 2-EH is also used as a solvent, a lubricant and as a finishing compound for paper and textiles. The preferred route of administration for the carcinogenicity studies was oral via the diet. However, feed blends containing neat 2-EH were not sufficiently stable for feed studies. Dosed feed blends prepared with neat 2-EH retained only 86% of the theoretical concentration after blending, and 46% of theoretical after storage for 2 days in a rat cage environment. Feed blends containing microencapsulated 2-EH were sufficiently stable for toxicity studies: no losses of 2-EH were observed after blending, feed blends stored for 7 days in a rat cage retained 99% of the theoretical concentration and blends stored in sealed containers at room temperature for 21 days retained 97% of the theoretical concentration. These studies demonstrate the potential for microencapsulation technology to eliminate dose formulation problems associated with volatile chemicals.

Comparison of the toxicity of citral in F344 rats and B6C3F1 mice when administrated by microencapsulation in feed or by corn-oil gavage.

A study of the potential effects of microencapsulation on the toxicity of citral was conducted in 14-day continuous feeding studies with both sexes of F344 rats and B6C3F1 mice. Toxicity by the feeding route was compared with that from bolus doses of the neat chemical in corn oil administrated by gavage. Both sexes of rats and mice were given diet containing 0, 0.63, 1.25, 2.5, 5 and 10% citral microcapsules. These feed formulations were equivalent to daily doses of 0, 142, 285, 570, 1140 and 2280 mg citral/kg body weight for rats and 0, 534, 1068, 2137, 4275 and 8550 mg citral/kg body weight for mice. The daily gavage doses were 0, 570, 1140 and 2280 mg citral/kg body weight for both sexes of rats, and 0, 534, 1068 and 2137 mg citral/kg body weight for both sexes of mice. Citral microcapsules administered in the diet did not cause mortality in mice or rats. Toxicity was confined to decreases in body weight at the 10% concentration in mice, at the 5 and 10% concentrations in rats, and decreases in absolute weights of the liver, kidney and spleen at the 10% concentration in rats. The only histopathological change observed was minimal to mild hyperplasia and/or squamous metaplasia of the respiratory epithelium in the anterior portion of the nasal passages of rats fed 5 or 10% citral microcapsules. By contrast, citral gavage caused mortality in five out of five male and female mice at 2137 mg/kg body weight, and in two out of five male mice at 1068 mg/kg body weight. There were dose-related increases in absolute liver weights of male and female mice. Cytoplasmic vacuolization of hepatocytes occurred in all female mice gavaged with 1068 and 2137 mg citral/kg body weight, and in male mice from the 2137 mg/kg dose group. Necrosis, ulceration and/or acute inflammation of the forestomach occurred in the high-dose mice of both sexes. Inflammation and/or hyperplasia of the forestomach occurred in about half of the male and female mice dosed with 1068 mg citral/kg. Citral gavage at doses that were equivalent to up to 10% in the diet (2280 mg/kg body weight) did not cause toxicity in rats, except for minimal hyperplasia of the squamous epithelium of the forestomach in high-dose males.(ABSTRACT TRUNCATED AT 400 WORDS)

Application of microencapsulation for toxicology studies. III. Bioavailability of microencapsulated cinnamaldehyde.

The bioavailability of microencapsulated cinnamaldehyde (CNMA) was investigated in male F344 rats. Rats were gavaged with CNMA in corn oil using either microencapsulated or the neat chemical at doses of 50, 250, and 500 mg/kg. No differences between the two formulations at any of the doses were found in either CNMA blood concentration profiles or in the rate of urinary hippuric acid excretion. Both formulations showed a low bioavailability (< 20%) at 250 and 500 mg/kg. Regardless of the formulation used, oral gavage of CNMA significantly increased the urinary excretion of hippuric acid. About 75% of the dose of CNMA was metabolized to hippuric acid and recovered in the urine. The total amount of hippuric acid recovered in a 50-hr urinary collection correlated well with the CNMA dose. The data suggest that there was complete release of CNMA from the microcapsules and that microencapsulation of CNMA does not affect its bioavailability or its metabolism. Since CNMA microcapsules are stable in rodent diet, the microencapsulation of CNMA, and perhaps other labile chemicals, will prevent degradation and facilitate the testing of such compounds in toxicology studies.

Toxicokinetics of cinnamaldehyde in F344 rats.

The toxicokinetic profile of cinnamaldehyde (CNMA) was investigated in Fischer 344 rats. CNMA was found to be unstable in blood. After iv administration, a large fraction of CNMA was immediately oxidized to cinnamic acid. The biological half-life of CNMA after iv administration was found to be 1.7 hr. After administration by gavage of CNMA at 250 or 500 mg/kg body weight using corn oil as vehicle, the maximum blood concentrations of CNMA were in the order of 1 microgram/ml. These low blood concentrations were maintained over a 24-hr period after a dose of 500 mg/kg, which is relatively long considering the short (1.7 hr) biological half-life of CNMA. The estimated oral bioavailability of CNMA was less than 20% for both the 250 and 500 mg/kg doses. No CNMA was present in blood at any time in rats dosed with 50 mg CNMA/kg body weight. Only a small amount of the administered CNMA was excreted in rat urine as free cinnamic acid or beta-glucuronide-conjugated cinnamic acid. The majority of CNMA administered orally was excreted in urine as hippuric acid within 24 hr. The maximum excretion rate occurred at 8 hr after gavage. Hippuric acid recovered in 50-hr urine samples was found to be directly proportional to the oral dose of CNMA.

Quantitation of cinnamaldehyde and cinnamic acid in blood by HPLC.

A rapid and sensitive high performance liquid chromatographic (HPLC) method is described for the quantitation of cinnamaldehyde (CNMA) in rat blood at concentrations of 0.1-100 micrograms/mL. One of the metabolites of CNMA, cinnamic acid, can also be quantified simultaneously. CNMA is unstable in rat blood, probably because of rapid oxidation to cinnamic acid by enzymatic catalysis and nonenzymatic Schiff base formation with free amine groups of blood proteins. The disappearance of CNMA from rat blood follows first-order reaction kinetics with a half-life of 9 min at room temperature. The current analysis method involves the addition of an agent that will prevent CNMA degradation by denaturing protein and competitively blocking nucleophilic addition reactions, resulting in the nearly complete recovery of CNMA from blood. Recovery of cinnamic acid was approximately 80% at concentrations of 1-10 micrograms/mL.

Development of renal toxicity in F344 rats gavaged with mercuric chloride for 2 weeks, or 2, 4, 6, 15, and 24 months.

Both sexes of F344 rats were gavaged with maximal tolerated doses of mercuric chloride for periods from 2 wk to up to 2 yr to investigate chronic nephrotoxicity and potential carcinogenicity. The toxicity of mercuric chloride was excessive after 2 wk of exposure to doses ranging from 1.25 to 20 mg/kg, compromising renal function by selectively destroying cells of the proximal tubules, and eliciting marked elevations in urinary biomarker enzymes diagnostic for acute renal tubule necrosis. In the 2-wk studies, urinary alkaline phosphatase and aspartate amino-transferase were most sensitive to renal mercury toxicity among a panel of six enzymes, exhibiting twofold increases above controls at the 5.0 mg/kg dose, before changes in the other enzymes occurred. Urinary lactate dehydrogenase was the most responsive enzyme, with up to 11-fold increases in activity above controls. In response to mercuric chloride exposure of 5.0 mg/kg for 2-6 mo, the greatest and most persistent increases in elevation of urinary enzyme activities were exhibited by alkaline phosphatase and gamma-glutamyl transferase, which increased two-to threefold above controls. At this interval, the maximal severity of the renal lesions in both sexes of rats was graded as minimal to mild. Beyond 6 mo none of the urinary enzymes measured in this study was adequate as biomarkers of nephrotoxicity, although the severity of the renal lesions had progressed. Mercury accumulated in a dose-related fashion primarily in the kidney, and to a lesser extent in the liver. The severity of the renal lesions was increased by continued exposure to mercuric chloride, as tissue concentrations of mercury rose in proportion to dose. Mercuric chloride treatment for 2 yr clearly exacerbated the severity of the spontaneous nephrotoxicity prevalent in aging F344 rats. The excessive mortality that occurred in the male rats was probably due to a combination of these factors. No renal tumors were detected in rats, possibly because the potential for their development was reduced; however, direct tissue contact with mercury induced squamous-cell papillomas of the forestomach in both sexes.