Disposition of lead (Pb) in saliva and blood of Sprague-Dawley rats following a single or repeated oral exposure to Pb-acetate.

Biological monitoring for lead (Pb) is usually based upon a determination of blood Pb concentration; however, saliva has been suggested as a non-invasive biological matrix for assessing exposure. To further evaluate the potential utility of saliva for biomonitoring, the disposition of Pb was evaluated in whole blood (WB), red blood cells (RBC), plasma, parotid gland, bone, and saliva following either a single oral dose of 100mg Pb-acetate/kg body weight in rats or approximately 1-week after 5 sequential daily oral gavage doses of 1, 10, or 100mg Pb-acetate/kg/day. Saliva volume, pH, total saliva protein, and alpha-amylase activity were also determined. At specified times post-dosing groups of animals were anesthetized and administered pilocarpine to induce salivation. Saliva was collected, the animals were humanely sacrificed, and tissue samples were likewise collected, weighed, and processed for Pb analysis. Following a single dose exposure to Pb-acetate, Pb was detectable in all samples by 30 min post-dosing. For both the single and repeated dose treatments the concentration of Pb was highest in WB and RBC relative to plasma and saliva. However, the Pb rapidly redistributed (within 5-days post-treatment) from the blood into the bone compartment based on the substantial decrease in WB and RBC Pb concentration, and the concurrent increase in bone Pb following repeated exposure at all dose levels. Although there is clear variability in the observed Pb concentrations in plasma and saliva, there was a reasonable correlation (r(2)=0.922) between the average Pb concentrations in these biological matrices, which was consistent with previous observations. The single oral dose of Pb-acetate resulted in a decrease in salivary pH which recovered by 24h post-dosing and a decrease in alpha-amylase enzyme activity which did recover within 5-days of ceasing exposure. It is currently unclear what impact these slight functional changes may or may not have on Pb salivary clearance rates. These results demonstrate a feasibility to rapidly detect Pb in saliva and suggest that saliva may correlate best with plasma Pb concentration.

Development of a physiologically based pharmacokinetic model for ethylene glycol and its metabolite, glycolic Acid, in rats and humans.

An extensive database on the toxicity and modes of action of ethylene glycol (EG) has been developed over the past several decades. Although renal toxicity has long been recognized as a potential outcome, in recent years developmental toxicity, an effect observed only in rats and mice, has become the subject of extensive research and regulatory reviews to establish guidelines for human exposures. The developmental toxicity of EG has been attributed to the intermediate metabolite, glycolic acid (GA), which can become a major metabolite when EG is administered to rats and mice at high doses and dose rates. Therefore, a physiologically based pharmacokinetic (PBPK) model was developed to integrate the extensive mode of action and pharmacokinetic data on EG and GA for use in developmental risk assessments. The resulting PBPK model includes inhalation, oral, dermal, intravenous, and subcutaneous routes of administration. Metabolism of EG and GA were described in the liver with elimination via the kidneys. Metabolic rate constants and partition coefficients for EG and GA were estimated from in vitro studies. Other biochemical constants were optimized from appropriate in vivo pharmacokinetic studies. Several controlled rat and human metabolism studies were used to validate the resulting PBPK model. When internal dose surrogates were compared in rats and humans over a broad range of exposures, it was concluded that humans are unlikely to achieve blood levels of GA that have been associated with developmental toxicity in rats following occupational or environmental exposures.

Development of a physiologically based pharmacokinetic model for propylene glycol monomethyl ether and its acetate in rats and humans.

Propylene glycol monomethyl ether (PM), along with its acetate, is the most widely used of the propylene glycol ether family of solvents. The most common toxic effects of PM observed in animal studies include sedation, very slight alpha(2u)-globulin mediated nephropathy (male rats only) and hepatomegally at high exposures (typically > 1000 ppm). Sedation in animal studies usually resolves within a few exposures to 3000 ppm (the highest concentration used in subchronic and chronic inhalation studies) due to the induction of metabolizing enzymes. Data from a variety of pharmacokinetic and mechanistic studies have been incorporated into a PBPK model for PM and its acetate in rats and mice. Published controlled exposure and workplace biomonitoring studies have also been included for comparisons of the internal dosimetry of PM and its acetate between laboratory animals and humans. PM acetate is rapidly hydrolyzed to PM, which is further metabolized to either glucuronide or sulfate conjugates (minor pathways) or propylene glycol (major pathway). In vitro half-lives for PM acetate range from 14 to 36 min depending upon the tissue and species. In vivo half-lives are considerably faster, reflecting the total contributions of esterases in the blood and tissues of the body, and are on the order of just a few minutes. Thus, very little PM acetate is found in vivo and, other than potential portal of entry irritation, the toxicity of PM acetate is related to PM. Regardless of the source for PM (either PM or its acetate), rats were predicted to have a higher Cmax and AUC for PM in blood than humans, especially at concentrations greater than the current ACGIH TLV of 100 ppm. This would indicate that the major systemic effects of PM would be expected to be less severe in humans than rats at comparable inhalation exposures.

Comparative metabolism of carbon tetrachloride in rats, mice, and hamsters using gas uptake and PBPK modeling.

No study has comprehensively compared the rate of metabolism of carbon tetrachloride (CCl4) across species. Therefore, the in vivo metabolism of CCl4 was evaluated using groups of male animals (F344 rats, B6C3F1 mice, and Syrian hamsters) exposed to 40-1800 ppm CCl4 in a closed, recirculating gas-uptake system. For each species, an optimal fit of the family of uptake curves was obtained by adjusting Michaelis-Menten metabolic constants Km (affinity) and Vmax (capacity) using a physiologically based pharmacokinetic (PBPK) model. The results show that the mouse has a slightly higher capacity and lower affinity for metabolizing CCl4 compared to the rat, while the hamster has a higher capacity and lower affinity than either rat or mouse. A comparison of the Vmax to Km ratio, normalized for milligrams of liver protein (L/h/mg) across species, indicates that hamsters metabolize more CCl4 than either rats or mice, and should be more susceptible to CCl4-induced hepatotoxicity. These species comparisons were evaluated against toxicokinetic studies conducted in animals exposed by nose-only inhalation to 20 ppm 14C-labeled CCl4 for 4 h. The toxicokinetic study results are consistent with the in vivo rates of metabolism, with rats eliminating less radioactivity associated with metabolism (14CO2 and urine/feces) and more radioactivity associated with the parent compound (radioactivity trapped on charcoal) compared to either hamsters or mice. The in vivo metabolic constants determined here, together with in vitro constants determined using rat, mouse, hamster, and human liver microsomes, were used to estimate human in vivo metabolic rates of 1.49 mg/h/kg body weight and 0.25 mg/L for Vmax and Km, respectively. Normalizing the rate of metabolism (Vmax/Km) by milligrams liver protein, the rate of metabolism of CCl4 differs across species, with hamster > mouse > rat > human.

The influence of ultrasound frequency and gas-body composition on the contrast agent-mediated enhancement of vascular bioeffects in mouse intestine.

The induction by ultrasound (US) of petechiae and hemorrhages in mouse intestine was examined with injection of gas body-based contrast agents. Production of petechiae in the intestinal wall was enhanced by contrast agents for both continuous and pulsed (10 micros pulses repeated at 1 kHz) exposure relative to a gas body-free blank. For pulsed exposure with 10 mL/kg of Albunex, apparent thresholds for peak negative pressure amplitude were 0.42 MPa at 0.4 MHz, 0.85 MPa at 1.09 MHz and 2.3 MPa at 2.4 MHz. Results at these frequencies were the same for 10-11 cycle pulses with fixed duty cycle (0.01). Thresholds for hemorrhage into the intestinal lumen were not appreciably enhanced by added Albunex, and appear to be compatible with previously reported lithotripsy data when duty factor differences are considered. The agents PESDA, Optison and Levovist had lower thresholds (for example, 1.8 MPa for Levovist) than Albunex at 2.3 MHz, and yielded more petechiae. The thresholds for petechiae induction by US with contrast agents encroach upon the exposure range relevant to diagnostic US practice.

Heating vs. cavitation in the induction of mouse hindlimb paralysis by ultrasound.

Grip strength tests were performed on hairless mice before and after various ultrasound exposures in a temperature-controlled water bath at 37 degrees C. Lithotripter exposure of 800 shock waves produced no effect on hindlimb function. In contrast, 1.09-MHz exposures at 1 MPa with 10:100 ms burst mode did produce a statistically significant reduction in grip strength of about 60%. The exposure duration was important for the 1.0-MPa burst mode exposure, with grip-strength reductions appearing after 150 s or longer exposures. Continuous exposure at 3.3 W cm(-2) (0.32 MPa peak) for 200 s produced the same effect as burst mode exposure at 3.3 W cm(-2) (1 MPa peak) for 200 s, which implicates the temporal average intensity as an important factor. The temperature elevations for 1-MPa burst mode was estimated from thermocouple measurements in the spine to be 12 degrees C after 200-s exposure. Although tests of exposures in cool (32 degrees C) and warm (42 degrees C) baths produced inconclusive results in regard to the thermal mechanism, the effects observed appear to result from ultrasonic heating (rather than from cavitation). Thus, any potentially harmful consequences associated with the effects examined might be related more, for example, to ultrasonic hyperthermia therapy than to shock-wave lithotripsy.

Consequences of lithotripter shockwave interaction with gas body contrast agent in mouse intestine.

PURPOSE: Shockwave lithotripsy can involve complications associated with hemorrhage and internal bleeding which appear to be due to acoustic cavitation. Gas-body-based contrast agents recently developed for diagnostic ultrasound can enhance cavitational bioeffects under some conditions. This study examined the occurrence and progression of vascular damage in mouse intestine when a contrast agent was present during shockwave treatment. MATERIALS AND METHODS: Anesthetized hairless mice were injected with Albunex contrast agent or a gas-body-free blank, and exposed to sham, 200 or 800 lithotripter shockwaves. RESULTS: Exposure of the mouse abdomen to lithotripter shockwaves produced petechiae in the intestinal wall and hemorrhage into the lumen. Contrast-agent gas bodies greatly enhanced the numbers of petechiae (but not the hemorrhages), relative to the blank agent. When evaluation of these effects was delayed for one day, both effects decreased, and the gas-body-associated petechiae seemed to disappear. However, survival significantly decreased for mice with added gas bodies and shockwave treatment. CONCLUSIONS: The presence of a gas-body-based ultrasound contrast agent enhances vascular side effects of shockwave lithotripsy. Although there are great uncertainties in relating these observations to human clinical conditions, a delay in planned treatment might be prudent for patients scheduled for shockwave lithotripsy soon after receiving gas-body-based ultrasound contrast agents.

Ultrasonic enhancement of gene transfection in murine melanoma tumors.

The enhancement of gene transfection by ultrasound (US) was evaluated in vitro and in vivo using the B16 mouse melanoma model. Cultured cells were either exposed in suspensions in vitro or implanted subcutaneously in female C57BL/6 mice for 10-14 days and, subsequently exposed, in vivo. For comparison to results with a luciferase plasmid, a reporter plasmid for green fluorescent protein (GFP) was used to evaluate transfection efficiency. US was supplied by a system, similar to a Dornier HM-3 lithotripter, that produced shock waves (SW) of 24.4 MPa peak positive and 5.2 MPa peak negative pressure amplitudes at the focus. The plasmids were mixed with the suspensions to achieve 20 ,microL mL(-1), or were injected intratumorally to provide 0.2 mg DNA per mL of tumor. Acoustic cavitation was promoted by retaining 0.2 mL of air in the 1.2-mL exposure chambers in vitro and by injecting air at 10% of tumor volume in vivo. In vitro, cell counts declined to 5.3% of shams after 800 SW exposure, with 1.4% of the cells expressing GFP after 2 days of culture. In vivo, 2 days after 400 SW exposure, viable-cell recovery from excised tumors was reduced to 4.2% of shams and cell transfection was enhanced by a factor of about 8, reaching 2.5% of cell counts (p < 0.005 in t-test). These results show that strong tumor ablation induced by US shock wave treatment can be coupled with simultaneous enhancement of gene transfection.

Gas-body-based contrast agent enhances vascular bioeffects of 1.09 MHz ultrasound on mouse intestine.

Anesthetized hairless mice were exposed to continuous or pulsed 1.09-MHz ultrasound with or without prior injection of a gas-body-based ultrasound contrast agent. Albunex at a dose of 10 mL/kg increased the production of intestinal hyperemia, petechia and hemorrhages by continuous ultrasound. For pulsed ultrasound, with 10 micros pulses and 0.01 duty cycle, petechiae were produced for exposures as low as 1 MPa spatial peak pressure amplitude with added gas bodies. The enhancement of petechiae production was robust for pulsed exposure; for example, at 2.8 MPa, an average of 227 petechiae was obtained with added gas bodies, which was 30 times more than without the agent. The production of petechia was roughly proportional to the dosage of Albunex for pulsed exposure. Results did not appear to be strongly dependent on pulsing parameters, but long bursts (0.1 s) were somewhat more effective than pulses (10 micros). The observed vascular bioeffects appeared to involve both thermal and nonthermal mechanisms for continuous exposure, but to result primarily from gas-body activation for pulsed exposure.

Enhancement of ultrasonically-induced hemolysis by perfluorocarbon-based compared to air-based echo-contrast agents.

Hemolysis induced by ultrasonic activation of various contrast-agent gas bodies was investigated. Canine whole blood, with high concentrations of the agents held in 1 mm thick chambers, was exposed in the nearfield of a 2.4-MHz ultrasound beam in a 37 degrees C water bath. Sterile phosphate buffered saline (PBS) served as a control agent without gas bodies. Albunex (Mallinckrodt Medical, St. Louis, MO) and Levovist (Schering AG, Berlin, Germany) represented the air-based contrast agents. The experimental agents FS069 (Optison, Molecular Biosystems Inc., San Diego, CA) and modified MRX-130 (ImaRx Pharmaceutical Corp., Tucson, AZ) represented perfluorocarbon-based contrast agents. No significant ultrasonically-induced hemolysis was detected for the PBS or Levovist suspensions. After 1 s continuous exposure, ultrasonically-induced hemolysis was significant for Albunex at 0.4 MPa or higher pressure amplitudes, for FS069 at 0.2 MPa and for modified MRX-130 at 0.4 MPa. Hemolysis found after pulsed exposure with 10 micros pulses and 1 ms pulse repetition period was significant for Albunex, FS069 and modified MRX-130 above thresholds of 1.1 MPa, 0.57 MPa and 1.6 MPa, respectively. FS069 led to more hemolysis after pulsed mode exposures of 1 s duration or longer than did Albunex. Reduced concentrations of gas bodies gave increased thresholds and reduced hemolysis. These results indicate that improvements in persistence of contrast agents, which increase their clinical utility, may also enhance the potential for cavitational bioeffects.

The interaction of ultrasonic heating and cavitation in vascular bioeffects on mouse intestine.

Anesthetized hairless mice were exposed at the midsection to 400-kHz focused ultrasound that was continuous or pulsed with 100 microseconds pulses, in a temperature-controlled water bath. After exposure, the intestines were evaluated for petechiae, presumably induced by heating, and hemorrhages, presumably induced by cavitation. Petechiae (up to about 100) occurred above 0.28 MPa (2.6 W cm-2) for 1000 s continuous exposure at 37 degrees C, and the threshold increased to 6.5 MPa (1.4 W cm-2 temporal average) for 1000 s pulsed exposure (0.001 duty factor). Hemorrhages (up to about 10) were seen above 0.65 MPa for continuous exposure (10 s, 100 s or 1000 s), and the threshold increased only to 1.6 MPa for 1000 s pulsed exposure (0.001 duty factor). Fractionating a brief continuous exposure into a low duty-factor pulsed exposure greatly decreased the petechiae, but actually increased the hemorrhages. For example, 1 s continuous exposure at 3.2 MPa (340 W cm-2) averaged 0.33 hemorrhages per mouse, and 1 s on-time pulsed exposure (1000 s duration, 0.001 duty factor) at 3.2 MPa (0.34 W cm-2 temporal average) averaged 4.3 hemorrhages per mouse. More petechiae were induced at 42 degrees C bath temperature relative to 32 degrees C or 37 degrees C, and the hemorrhage effect was somewhat enhanced by elevated temperature. Generally, heating and cavitation appeared to have largely independent roles in vascular bioeffects on mouse intestine.

In vivo transfection of melanoma cells by lithotripter shock waves.

The potential for gene transfection during shock wave tumor therapy was evaluated by searching for shock wave-induced DNA transfer in mouse tumor cells. B16 mouse melanoma cells were cultured by standard methods and implanted s.c. in female C57BL/6 mice 10-14 days before treatment. A luciferase reporter vector was used as the DNA plasmid for intratumoral injection at 0.2 mg/ml tumor. Air at 10% of tumor volume was injected after the DNA in some tumors to enhance acoustic cavitation activity. The shock wave generation system was similar to a Dornier HM-3 lithotripter with pressure amplitudes of 24.4 MPa peak positive and 5.2 MPa peak negative. Luciferase production in isolated tumor cells was measured with a luminometer 1 day after treatment to assess gene transfer and expression. Exposure to 800 shock waves, followed by immediate isolation and culture of tumor cells for 1 day, yielded 1.1 (0.43 SE) pg/10(6) cells for plasmid injection only and 7.5 (2.5 SE) pg/10(6) cells for plasmid plus air injection. Significantly increased luciferase production, relative to shams, occurred for 200-, 400-, 800-, and 1200-shock wave treatments with plasmid and air injection. Exposure with the isolation of tumor cells delayed for a day to allow gene expression within the growing tumors gave increased luciferase production for 100- and 400-shock wave exposures without and with air injection. Gene transfer therefore can be induced during lithotripter shock wave treatment in vivo, particularly with enhanced acoustic cavitation, which supports the concept that gene and shock wave therapy might be advantageously merged.

Induction of micronuclei in respiratory tract following radon inhalation.

Male Wistar rats were exposed to radon and its progeny (0.0, 60, 262 and 564 working level months, WLM), and the frequency of micronuclei was determined in deep lung fibroblasts, and deep lung, trachea and nasal epithelial cells with slopes of 0.28, 0.67, 0.34 and 0.11 micronuclei/1000 binucleated cells/WLM respectively. Micronuclei in deep lung fibroblasts, isolated and cultured using two methods and media, demonstrated no differences in slopes. Biological damage was used as a biodosimeter to calculate the relationship between dosimetric units: alpha particle traversals or 'nuclear hits', dose in mGy and exposure in WLM. The estimated number of nuclear alpha traversals/Gy was 6.3. Radon exposure to 170 WLM resulted in the same frequency of micronuclei in deep lung epithelial cells as produced by one alpha hit/cell nucleus. Absorbed dose/unit of exposure (mGy/WLM) was estimated assuming the damage was related to absorbed dose or to changes in cell sensitivity and ranged from 1.13 to 1.34 for deep lung epithelial cells, 0.47 to 1.09 for deep lung fibroblasts, 0.34 to 0.67 for tracheal epithelial cells and 0.18 to 0.33 for nasal epithelial cells. Biological dosimetry can be used to relate exposure to damage, compare dosimetric units and validate physical dosimetry models. This approach can be applied to any inhaled material capable of producing biological damage.

Ultrasonically induced hemolysis at high cell and gas body concentrations in a thin-disc exposure chamber.

Ultrasound image contrast may be enhanced by injecting gas bodies into the blood. This in vitro study was undertaken to assess the potential for induction of hemolysis due to ultrasonic activation of the contrast agent gas bodies. Canine whole blood with Albunex (Mallinckrodt Medical, St. Louis, MO, USA) was exposed to near-field ultrasound beams in 1-mm-thick chambers held stationary (i.e., not rotated) in a 37 degrees C water bath. At 2.25 MHz, statistically significant hemolysis occurred in 0.5 hematocrit, 50% Albunex suspensions for 0.28-MPa, 1-s continuous exposure and for 0.58-MPa, 100-s exposures with 10-microsecond pulses and 1.0-ms pulse repetition period. Continuous exposure durations as short as 10 ms produced about 4.5% hemolysis, which only increased slightly to about 5.5% after 100 s. At a constant 1.6 MPa, hemolysis increased with increasing gas body concentration and with decreasing cell concentration. Hemolysis decreased with increasing frequency in a 50/50 mixture of whole blood and Albunex, with thresholds rising from 0.12 MPa continuous (1 s) and 0.47 MPa pulsed (10 microseconds:1.0 ms for 100 s) at 1.06 MHz to 0.47 MPa continuous and 1.9 MPa pulsed at 5.3 MHz.

Deposition and early disposition of inhaled 233UO2(NO3)2 and 232UO2(NO3)2 in the rat.

Little information exists on the metabolism and potential health effects of 233U and 232U, high-specific-activity U isotopes associated with Th breeder systems. This paper describes the distribution and retention of the two isotopes following inhalation of uranyl nitrate, a simulated process solution. The lungs of rats exposed to 233UO2(NO3)2 and 232UO2(NO3)2 aerosols contained from 7 to 23% of the total amount of U retained in the rat after a 30-min inhalation exposure. Uranium was translocated rapidly from the lung and was retained mainly in skeleton, kidney and liver. Amounts equivalent to from one-quarter to one-half the initial lung burden (ILB) of U were excreted in urine the first day after inhalation. Radiation dose estimates based on 233U and 232U retention kinetics indicate that lung and skeleton would be the target organs for delayed radiation effects.