* Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, and
Section of Environmental Toxicology, GSF-Institut für Toxikologie, Neuherberg, Germany
Received April 29, 2003; accepted August 1, 2003
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ABSTRACT |
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Key Words: chloroacetic acid; chloroacetate; oral toxicity; dermal toxicity; low dose kinetics; high dose kinetics; oral absorption and sequestration; dermal absorption and sequestration; therapeutic inferences.
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INTRODUCTION |
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MCA is rapidly absorbed through the skin and GI tract. It is not only highly corrosive to tissues, but can cause death following dermal exposure as well as after ingestion. There have been a number of reports in the literature on accidental poisoning of humans and animals, mostly with fatal outcomes (Kulling et al., 1992; Kusch et al., 1990
; Mann, 1969
; Millischer et al., 1987
; Quick et al., 1983
; Rogers, 1995
; Zeldenrust, 1951
). The intensity of the acute toxicity of MCA is apparent from the fact that hospitalization has been recommended for individuals having exposure to as little as 1% of the skin to technical grade formulations (Kulling et al., 1992
; Kusch et al., 1990
).
The exact mechanism of MCA induced toxicity is not known. However, MCA reportedly interferes with ATP formation in the tricarboxylic acid (TCA) cycle and gluconeogenesis (Doedens and Ashmore, 1972; Fuhrman et al., 1955
; Gosselin et al., 1984
; Hayes et al., 1973
; Kulling et al., 1992
). MCA has also been reported to reduce sulfhydryl content in liver and kidney (Fuhrman et al., 1955
; Gosselin et al., 1984
; Hayes et al., 1973
). These effects facilitate severe tissue damage in organs such as liver, heart, CNS, kidney, and skeletal muscle (Hayes et al., 1973
; Kulling et al., 1992
).
The acute, subchronic, and chronic toxicities including a lack of mutagenic and carcinogenic potency of MCA have been thoroughly investigated (Bhat et al., 1991; Bryant et al., 1992
; Davis and Bernt, 1987
; Hayes et al., 1973
; Innes et al., 1969
; NTP, 1992
; Rannug et al., 1976
; van Duuren, 1974
). However, information on the kinetic profile of MCA is limited (Bhat et al., 1990
; Kaphalia et al., 1992
). Earlier, this laboratory has reported the kinetic profile of MCA in rats following iv administration (Saghir et al., 2001
). This study describes the kinetic profile of MCA in rats after oral (subtoxic and toxic doses) and dermal (toxic dose) administration.
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MATERIALS AND METHODS |
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Animals.
Adult male Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and allowed to acclimatize to the animal facility for one week before their use in experimentation. Animals were housed in polycarbonate cages (three per cage) with corncob bedding and stainless steel wire tops under standard conditions (22 ± 1°C, 55% relative humidity, 12-h light/dark cycle). Rats were provided with Teclad 7001 Rodent Chow (Harlan, Madison, WI) and water ad libitum, and were not fasted before dosing. After acclimatization, animals (160170 g) were randomly selected for experimentation.
Oral Administration
Dose formulation and administration.
The oral dose solutions (5 and 112.5 mg/ml) were prepared in HPLC grade water and used without adjusting the pH in order to simulate actual accidental poisoning scenarios. Dose solutions were kept in the dark at 4°C and brought to room temperature before administration. Rats were orally dosed (gavaged) with an acutely subtoxic (10 mg/kg) dose or an acutely toxic (225 mg/kg, ~LD20) dose of MCA at a volume of 2 ml/kg (15.0 µCi/kg) using a glass disposable syringe and disposable stainless steel feeding needle. After completion of dosing, rats were transferred to individual metabolism cages. Animals had free access to food and water throughout the course of the study.
Dose selection.
To select the acutely toxic dose of MCA, groups of five rats were exposed to non-radiolabeled MCA at doses ranging from 75 to 500 mg/kg. The onset of toxicity (coma and mortality) was closely monitored for up to 72 h after the administration of MCA. On the basis of the acute toxicity results, rats were dosed with an acutely subtoxic dose of 10 mg/kg or an acutely toxic (~LD20) dose of 225 mg/kg MCA for the oral kinetic study.
Sample collection and analysis.
Three rats were euthanized by decapitation at various time intervals (0.25, 0.75, 2, 4, 8, 16, and 32 h) after dosing. Due to expected mortality at the toxic dose, four additional animals were dosed in order to have at least three surviving rats at each time-point of termination. Blood was collected from cervical stumps in tubes containing EDTA and separated into plasma and red blood cells by centrifugation. Urinary bladders were manually emptied and the collected liquids added to the appropriate urine samples. Various organs (liver, kidney, heart, brain, thymus, fat, brown fat, lung, testis, spleen, muscle, skin [ear], and intestine) were removed from each animal by gross dissection. The gastrointestinal (GI) tract was dissected into stomach (esophagus and stomach), small intestine and large intestine and contents procured. The collected tissues and contents were processed as described earlier (Saghir et al., 2001). The same tissues as described above were also collected from rats that died before their scheduled sacrifice. Total radioactivity in the collected samples was quantitated in appropriate aliquots as described by Saghir et al.(2001)
. Background radioactivity from tissues and fluids of a rat not given 14C-MCA was subtracted from each sample before calculating concentrations.
Kinetic analysis.
Average plasma concentration-time profiles of total radioactivity was analyzed by both noncompartmental and compartmental modeling methods using a nonlinear least square regression program (WinNonlin, Pharsight Corp., Cary, NC) with first-order input, first-order output and no lag time of absorption. Noncompartmental methods were used to determine AUC032, AUC0
, and mean residence time (MRT) and a compartmental model to obtain estimates of absorption (Ka), peak blood concentration (Cmax), and the time to maximum concentration (Tmax). Bioavailabilities were calculated from the ratios of the AUC0
with that of the iv route (Saghir et al., 2001
). The time course of 14C-MCA in selected tissues (liver, kidney, heart, brain, stomach, and small intestine) was also analyzed in terms of kinetics to determine rate constants for uptake and removal as well as AUC0
32, AUC0
, Cmax, Tmax,, and MRT using compartmental and noncompartmental methods.
Metabolites.
Diluted samples of small intestinal contents were centrifuged and analyzed for parent MCA and total metabolite(s) using HPLC as described earlier (Saghir et al., 2001). Metabolic profile of MCA in blood, urine, and bile has been described after iv injection (Saghir et al., 2001
) and were therefore not evaluated in this study.
Statistics.
14C-MCA concentrations found in selected tissues of the rats that died at unscheduled times were statistically compared to those found in tissues of rats euthanized within the time window of death using Students t-test. Data in text, tables, and figures are given as mean ± SE.
Dermal Administration
Dose formulation and application.
The dermal dose solution (250 mg/ml) was prepared in acetone, and pH of the dose solution was not adjusted in order to simulate actual accidental dermal poisoning scenarios in humans. Dose solution was kept in the dark at 4°C and brought to room temperature before administration. Rats were dermally administered an acutely toxic (125 mg/kg, ~LD20) dose of MCA at a volume of 0.5 ml/kg (6.4 µCi/kg). Briefly, dermal application of MCA proceeded with careful shaving of an area of about 34 cm2 from the back of each rat, slightly posterior to the scapulae. Rats having nicks or cuts on the skin were excluded from the study. Animals were dosed topically with MCA in acetone (within an area of about 1 cm in diameter) in the conscious state. The dose solution was applied evenly to the skin using a round-tipped feeding needle attached to a disposable glass syringe. The dosing site of the animals was thereafter covered with a piece (~2 cm2) of parafilm and secured with Conform adhesive bandage (Kendall Medical Products, Mansfield, MA). After completion of dosing, rats were transferred to individual metabolism cages. Animals had free access to food and water during the entire course of the study.
A group of 21 rats was also dosed dermally with a subtoxic dose of 10 mg/kg the same way described above, except that the skin at the dosing site was covered with cotton gauze before securing with adhesive bandage. Later, it was discovered that 2046% of the applied dose migrated into the gauze and therefore was unavailable for dermal absorption. The method was modified for the toxic dose by covering the dosing site with parafilm and securing it with an adhesive bandage. The subtoxic dose experiment was not repeated and the results are not considered suitable for reporting.
Dose selection.
Similar to the oral dose selection, the toxic dermal dose was selected by exposing groups of five rats to non-radiolabeled MCA at doses ranging from 75175 mg/kg and closely monitoring the onset of toxicity (coma and mortality) for up to 72 h after the dermal application of MCA. On the basis of these results, an acutely toxic dose of 125 mg/kg (~LD20) was selected for the dermal kinetic study.
Sample collection and analysis.
In addition to the collection and processing of samples described above, parafilm covering the dose site was saved from the dermally dosed rats. The skin at the application site was thoroughly wiped with several damp cotton gauze to determine remaining dose at the site of application. Skin at the dosing site was also excised to measure sequestered radioactivity therein. Dose site wipes and parafilm coverings were extracted with water and radioactivity determined in aliquots as described by Saghir et al.(2001).
None of the sample from the dermally dosed rats was analyzed for metabolite(s). Kinetic and statistical analysis of the data obtained from the dermal study was similar to that described above for the oral dose.
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RESULTS |
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Absorption.
Figure 1 shows disappearance of 14C-MCA from stomach after oral gavage. The subtoxic dose (10 mg/kg) disappeared rapidly leaving <35 and 10% of radioactivity after 0.25 and 0.75 h, respectively (Table 1
). Disappearance of the subtoxic dose from stomach followed apparent first-order kinetics (Fig. 1
). The toxic dose of 225 mg/kg caused the stomach to distend profoundly with fluid (probably due to increased osmolarity) restricting the movement of stomach contents into the small intestine. This was observed in all rats sacrificed between 0.75 and 8 h. Within the first 0.25 h, 37% of the toxic dose was absorbed from stomach and/or moved into the small intestine as compared to 82% of the subtoxic dose during the same time period (Tables 1
and 2
). Absorption of the toxic dose from the stomach was saturated between 0.25 and 8 h and followed apparent zero-order kinetics probably due to the lack of movement of stomach contents into the small intestine. Severe restriction of stomach emptying was quite apparent as only 35% of the administered toxic dose reached the small intestine during 4 h after dosing after the initial burst of absorption (Table 2
). High but decreasing concentrations were found in the stomach wall compatible with continuous absorption from the stomach as well as increasing fluid content, which also contributed to decreasing concentrations in the stomach contents. In contrast, the subtoxic dose rapidly moved into the small intestine (>20 and 45% of dose was recovered from the small intestine within 0.25 and 0.75 h after dosing, respectively) and was quickly absorbed from there (Table 1
). After the initial rapid absorption of the released fraction of the toxic dose (within 0.25 h of dosing), only an additional 8 and 26% of dose was absorbed during the next 4 and 8 h, respectively, which was consistent with the presence of 55 and 37% of the dose in stomach contents 4 and 8 h after dosing (Table 2
). Absorption of either dose was virtually complete from the GI tract as only a maximum of 1.5% of dose was eliminated through feces (Tables 1
and 2
).
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Metabolism.
Most of the radioactivity recovered from the small intestine of rats dosed orally was parent MCA, representing more than 90% of radioactivity 0.25 h after the administration of the subtoxic dose and declining to 67% by 4 h (Table 3). This was an indication of biliary excretion and possibly enterohepatic circulation of (a) metabolite(s). In rats dosed orally with the toxic dose, 7686% of the recovered radioactivity from the small intestine was parent MCA (Table 3
). There is an apparent increase in the amount of parent MCA in the small intestine at later time points (832 h) when the total amount present became quite small (Tables 1
, 2
, and 3
). Rats that died at unscheduled times after the toxic dose had only 46% of parent MCA in their small intestine compared to 80% in rats sacrificed 0.75 and 2 h after dosing (the time frame of unscheduled death; Table 3
). Metabolic profiling of MCA in bile, urine, and plasma of rats was not conducted as it has been already reported after iv administration (Saghir et al., 2001
).
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Comparison of tissue levels between rats that died at unscheduled times and those sacrificed as scheduled.
Table 4 compares the tissue concentration of 14C-MCA in rats that died as a result of toxicity before their scheduled termination with those sacrificed at the two closest experimental time points. Mortality occurred 1.7 ± 0.2 h after oral dosing and thus, levels of radioactivity in tissues of rats found dead were compared with those sacrificed at 0.75 and 2 h after dosing. Statistically significantly higher concentrations of radioactivity were found in blood, lungs, muscles, and most other organs of the rats which died during the experiment when compared to tissue levels of rats which were sacrificed 2 h after oral dosing. This is compatible with more efficient absorption of MCA in these animals as also indicated by lower residual radioactivity in stomach and lower concentrations of parent MCA in the small intestine (Tables 3
and 4
).
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The MRT of the subtoxic dose in both stomach and small intestine was about 2 h, whereas MRT of the toxic dose in the small intestine was about 11 h (Table 6). Differences between tissue AUCs at the subtoxic and toxic doses were in the expected range of about 20-fold, representing the quotient of doses, with the exception of the heart and brain, which were 4355 times higher at the toxic dose (Table 6
). Modeled estimates of Tmax agreed with data observed in most tissues (Tables 1
, 2
, and 6
).
Dermal Administration
Acute toxicity.
The dose-response for toxicity (coma and death) of MCA after a single dermal application was also very steep with no apparent signs of toxicity up to 100 mg/kg and 21% mortality at 125 mg/kg. Mortality increased to 60% at 150 mg/kg and all of the treated rats died at 175 mg/kg. Similar to the oral dosing, mortality was preceded by coma in all cases and accompanied by clonic and tonic convulsions. In most instances animals died within minutes after the onset of coma and none of the comatose rats survived. The LD50 after a single dermal application was calculated to be 145 mg/kg. It took longer to induce coma and death after dermal application of the toxic doses compared to the rats dosed orally; mean time to coma and death was 2.4 ± 0.1 h (n = 12) and 2.8 ± 0.1 h (n = 12), respectively. On the basis of the results of acute toxicity, 125 mg/kg (~LD20) was chosen as the toxic dose for the dermal kinetic study.
Absorption.
The dermally applied 14C-MCA rapidly penetrated into the skin leaving only 1.8 ± 0.3% of the applied dose at the site of application by 0.25 h (Fig. 3). Only three out of 21 rats had elevated residual MCA (511% dose) at the site of application. Acidity of the dosing solution caused some erosion of the epidermis at the dosing site. Significant fractions of the penetrated MCA remained sequestered in deeper skin layers at the dosing site for an extended period of time and were slowly absorbed from there; about 50 and 20% of the dose was still trapped in the dermis 0.75 and 4 h after dosing, respectively. Seven percent of the dose was still present in the skin at the application-site 32 h after dosing (Fig. 3
).
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Excretion.
Dermally applied MCA was also excreted rapidly, with 8% of dose appearing in the urine within 2 h. Urinary excretion of MCA reached 21% of dose by 4 h, with total recovery amounting to 64% of dose by 32 h after dosing (Table 7). Similar to the oral route of exposure, fecal excretion of radioactivity in dermally dosed rats was very limited compared to the amount found in the GI tract; a total of only about 1% of dose was recovered in feces (Table 7
).
Comparison of tissue levels between rats that died at unscheduled times and those sacrificed as scheduled.
Mortality occurred about 3 h after the dermal application of MCA and therefore, tissue concentrations of dead rats were compared with those sacrificed at 2 and 4 h after dosing. Concentrations of radioactivity in tissues of rats that died before scheduled sacrifice were either identical or lower than those sacrificed at 2 or 4 h with the exception of kidney, which was significantly higher than in rats sacrificed 2 h after dermal dosing (p < 0.01). Concentrations of MCA trapped in the dermis at the dosing site was statistically not different between rats that died of intoxication before scheduled sacrifice and those sacrificed on schedule (Table 4). Also, there was no clear trend in the concentration profile between the two groups of rats.
Kinetic analysis.
14C-MCA was rapidly absorbed (Ka = 1.8 ± 0.3 h-1) from the application site without any apparent lag time (Fig. 4). Dermal bioavailability was >90% (Table 5
). The MRT of 14C-MCA in plasma was 6.4 h. The model predicted a Cmax of about 61 µg/ml-1, which was achieved 1.4 h after application (Table 5
). Plasma elimination half-life of total radioactivity was 3.7 ± 0.2 h and total body clearance was 267.5 ± 16.7 ml h-1 kg-1. The AUC0
32 was 447 ± 25 µg h ml-1, which was increased to 462 ± 28 µg h ml-1 when extrapolated to infinity (AUC0
). A one-compartmental model provided a better fit for the plasma data than a two-compartment model, predicted values being very close to those observed (Fig. 4
). During the last 16 h (1632 h after dosing) plasma concentration of 14C-MCA declined only by ~1 µg/ml-1, whereas the model predicted a decline of 4 µg/ml-1 (Fig. 4
). The kinetic model used could not account for constant influx of MCA from the dermis at the dosing site for up to 32 h and therefore predicted an earlier and hence steeper decline.
The dermally applied dose was rapidly shunted to the liver with an uptake rate constant of 9 ± 5 h-1. Very fast distribution of MCA to the liver was also apparent from the appearance of a large amount of radioactivity in the small intestine which could have occurred only via bile (Table 7) with a high appearance rate constant (1.2 ± 0.4 h-1; Table 6
). The rate constant for removal of radioactivity from the liver was slower than expected (0.07 ± 0.01 h-1), probably due to enterohepatic circulation (Table 7
) or trapping of MCA in the TCA cycle. Distribution of 14C-MCA to the brain was slower than to the heart (0.75 versus 1.2 h-1, respectively). MCA was eliminated from brain very slowly (0.04 h-1); the brain retaining high MCA levels for the longest period of time (Table 7
). Interestingly, kidney had an identical rate constant for uptake and removal of radioactivity (0.23 h-1) indicating efficient renal clearance and a lack of reabsorption of MCA from kidney, compatible with its PKa. A one-compartment model provided the best fit to tissue data.
The MRT of the dermally applied 14C-MCA in liver, kidney and heart was 8 h and in the small intestine it was 6 h (Table 6). Similar to the other routes (oral and iv), MRT of 14C-MCA in brain was longer than in any other tissue. The rank order was iv >> dermal
oral (Tables 2
, 6
, and 7
; Saghir et al., 2001
). Model estimates for Tmax agreed with observed data for all tissues examined with the exception of liver, which was predicted to reach Cmax within an hour but actually peaked at 4 h after dermal application (Tables 6
and 7
).
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DISCUSSION |
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Rapid absorption of MCA from the GI tract was expected because of the quick onset of coma and death after toxic doses. This is also consistent with the reported fatal poisoning case of a 5-year-old girl who was mistakenly given one teaspoon of MCA instead of a cold syrup. She died 8 h after the ingestion of MCA (Feldhaus et al., 1993; Rogers, 1995
).
The gross distension of stomach of rats dosed with the toxic dose of 225 mg/kg and the restriction of the flow of stomach contents to the small intestine was not anticipated. Rapid initial transfer into the small intestine is clearly documented by plasma data, decreased stomach contents, and increased small intestinal concentrations of MCA. It appeared that irritation of the pylorus between 0.25 and 0.75 h after dosing led to a spasm of the sphincter, which prohibited further flux for several hours. During this time, water was drawn into the stomach due to MCA-induced high osmolarity. Increased water content reduced the concentration of MCA in the stomach, which in turn resulted in reduced irritation and eventually in the opening of the pylorus, after about 8 h.
Rapid initial absorption followed by an extended period of slow zero-order absorption from the stomach mimics kinetically a loading dose and subsequent iv infusion, which leads to virtual steady-state plasma concentration during the first 2 h (Table 2). This "ideal" kinetic condition results in a steep dose response. Virtual steady-state concentration of MCA is also consistent with the acute toxicity finding that none of the animals recovered from coma at the higher toxic doses of
350 mg/kg and died within minutes after the onset of coma. This was not the case with iv injections (Saghir et al., 2001
) or in animals receiving less toxic oral doses (between 225300 mg/kg), which remained in deep stupor for extended periods of time and some of them recovered without showing any further signs of toxicity.
A rapid distribution of MCA has been reported after iv dosing (Saghir et al., 2001) as expected since MCA is a very small (molecular weight = 94.5) water-soluble molecule, which moves into and out of body compartments with bulk flow of water (Saghir et al., 2001
). Similar to the earlier findings, higher levels of radioactivity were found in most of the richly perfused tissues including liver, kidney, heart, thymus, and brain. A higher concentration gradient between liver and other tissues at early time points and a larger rate constant of uptake (Tables 1
, 2
, and 6
) indicated active uptake of MCA by liver, which was also reported after iv dosing (Saghir et al., 2001
). The presence of metabolite(s) in the GI tract was an indication of the biliary excretion of MCA derivatives, similar to the earlier report (Saghir et al., 2001
). In addition, absorption of all the radioactivity from the GI tract and its negligible (<1% of dose) fecal elimination indicated enterohepatic circulation similar to what has been reported for iv injection (Saghir et al., 2001
). As reported, the major route of MCA elimination was via urinary excretion. An identical rate constant of uptake and removal of MCA from kidney (Table 6
) is compatible with no active uptake of MCA in kidney, a fact which has been also reported after iv administration (Saghir et al., 2001
). The total body clearance of MCA after toxic oral dose (558 mg h-1 kg-1; Table 5
) was higher than after the toxic iv injection (262 ml h-1 kg-1; Saghir et al., 2001
), which was certainly due to hepatic first-pass effect after oral administration.
Comparison of MCA levels in different tissues of rats that died before their scheduled termination with those sacrificed at approximately the same time suggested that death versus survival depended on the rate of absorption of MCA from the GI tract in nonsurvivors, overwhelming the detoxification capacity of the organisms. The time course of detoxification of MCA has been reported to be very similar to the time course of intoxication, both of them occurring concomitantly on a time scale of a few hours (Saghir et al., 2001). An indication of the detoxification is the total body clearance, which was lower in animals dosed with the toxic dose, suggesting a saturation of the detoxification processes (Table 5
). At the subtoxic dose of 10 mg kg-1, detoxification processes remained unsaturated after both oral and iv administration, indicated by higher and an almost identical total body clearance (769 versus 750 ml h-1 kg-1 for the oral and iv routes, respectively.
The insight gained from this oral kinetic study can be used to better understand the cause of death of the accidentally poisoned 5-year-old girl with MCA. Although the girl vomited immediately, she soon collapsed and died within 8 h (Feldhaus et al., 1993; Rogers, 1995
). The time lapse between the ingestion and vomiting, although not reported, appeared to be long enough to have rapid initial absorption of ~40% of the ingested MCA, similar to the rapid initial absorption of the toxic dose (within 0.25 h of dosing) in this study (Table 2
). Timing of emesis (within 0.25 h) is supported by the fact that the girl vomited immediately, at home, before her admission to the emergency department within 0.75 h postingestion (Feldhaus et al., 1993
). Assuming the girl weighed 18 kg (50th percentile weight for a 5-year-old girl) would yield an estimated dose of about ~430 mg/kg (specific gravity = 1.58 at 20°C). If 40% of this dose was absorbed rapidly and half of the remaining dose vomited, the systemically available dose would be about 300 mg/kg, which is in the range of a LD40 of this study and above the LD50 of earlier reported mouse studies (Morrison, 1946
; Woodard et al., 1941
). The residual levels of MCA found in the stomach/GI tract (~2337 mg, 30% of the dose) would have been enough to maintain high steady-state blood levels for many hours, depriving this child from recovery and eventually causing her death. This explanation is consistent with the high level of MCA found in her blood (100 µg/ml) even 8 h postingestion (Feldhaus et al., 1993
; Rogers, 1995
), about twice the amount as that found in plasma of rats 0.25 h after dosing and seven times higher than that found in plasma of rats 8 h after dosing with the toxic dose in this study (Fig. 2
).
In conclusion, low dose kinetics did not predict the high dose kinetics of MCA after oral administration as was also the case with iv injection.
Dermal Administration
MCA when applied dermally is intermittent in terms of acute toxicity between iv and oral dosing; the dermal LD20 (125 mg/kg) was two times higher than the reported LD40 (60 mg/kg) after iv injection (Saghir et al., 2001) and two times lower than the oral LD20 (225 mg/kg). Toxicity was in the order of iv > dermal > oral route of exposure. Higher toxicity of dermally applied MCA than after oral administration was due to its rapid absorption from the application site and sequestration of a large fraction of the applied dose in deeper skin layers (Fig. 3
). Rapid dermal absorption of MCA was expected because most of the reported toxicity in human patients occurred after dermal exposure. Other suggestive evidence was the fact that most of them died later in spite of rapid and vigorous washing having been started within minutes of exposure (Kulling et al., 1992
; Kusch et al., 1990
; Millischer et al., 1987
; Zeldenrust, 1951
). It is certainly plausible that damage to the epidermis (due to acidity) enhances penetration and increases dermal bioavailabilty of toxic doses.
Sequestration of MCA at the site of application in the skin created a "depot" causing a continuous influx of MCA as shown by constant plasma concentrations for up to 4 h (Table 7). This steady-state exposure of the animals during the time course of intoxication (2.8 ± 0.1 h) provides an explanation for obtaining a well-defined and very steep dose response (coma and death) after dermal application. A dose response for MCA was more difficult to establish after iv (Saghir et al., 2001
) and oral dosing and was less steep. The LD50 of the iv dose was between 70 and 100 mg/kg and of oral dose was >400 and <450 mg/kg, compared to a well-defined dose of 145 mg/kg after dermal application. Difficulty in establishing as good dose-response curves after oral and iv dosing was due to rapid detoxification of the administered MCA by liver resulting in falling plasma concentrations indicative of concurrently occurring intoxication and recovery particularly after iv dosing. A single iv dose actually measured the relationship between dose, toxicity, and recovery rather than between dose and toxicity alone because of the short half-life of MCA.
Sequestration (depot) of MCA in the skin mimicked continuous iv infusion, especially during the time-course of intoxication and prevented any recovery from occurring. The oral dose did not yield as good a steady state as the dermal application, although a better steady state than the iv injection. Accordingly, the oral dose response was steeper than the iv dose response but less steep than the dermal dose response. Concentration of 14C-MCA in plasma of the rats dosed dermally with 125 mg/kg was 28 times higher than those dosed orally with 225 mg/kg up to 4 h after dosing (Tables 2 and 7
). Slowed down absorption of the toxic oral dose and a protective hepatic first-pass effect allowed animals to recover faster which made the oral doses less toxic than iv and dermal doses. These findings are also consistent with results of the acute toxicity studies showing that none of the dermally dosed animals recovered from coma; all of them died soon after the onset of coma in contrast to the iv and oral routes of exposure. A much slower detoxification of dermally applied MCA is also apparent from reduced but sustained metabolism/biliary excretion, which in turn was due to its slow release from the storage depot in the skin (Table 7
). A continuous influx of MCA from this depot was also the reason for poor correlation between observed plasma levels of MCA and model predictions between 1632 h after dosing (Fig. 4
). The model predicted a sharp decline in plasma MCA levels in contrast to the observed flattening of the curve due to this continued influx, which the model could not accommodate.
Similar to the oral route of exposure, a rapid distribution of the dermally absorbed MCA to tissues was observed due to its high water solubility and low molecular weight. There was a 2- to 6-fold concentration gradient between liver and other tissues at early time points (up to 0.75 h; Table 7), indicating active uptake of MCA by the liver, similar to iv (Saghir et al., 2001
) and oral dosing. A 7- to 38-times larger rate constant of uptake by liver than by any other richly perfused tissue such as heart, brain, and kidney (Table 6
) further supports the view of active uptake.
The presence of high concentrations of radioactivity in the GI tract of the dermally dosed rats is again indicative of biliary excretion of MCA metabolite(s) similar to the finding after iv (Saghir et al., 2001) and oral dosing. Likewise, all of the 14C-MCA excreted with bile was reabsorbed from the GI tract through the enterohepatic circulation with negligible fecal elimination (Tables 1
, 2
, and 7
) after all three routes of exposure. As reported for the oral and iv dosing, the major route of MCA elimination was via urinary excretion after dermal administration. An identical rate constant of uptake and removal of MCA from kidney (Table 6
) is compatible with no active uptake of MCA in kidney, a fact which has also been observed after oral and iv administration. Total body clearance of the toxic dermal dose (268 mg h-1 kg-1) was identical to that calculated after the toxic iv injection (262 mg h-1 kg-1; Saghir et al., 2001
), an indication of similarity in the kinetics of dermally and iv administered MCA. The total body clearance after toxic oral administration was twice that of the iv and dermal routes of exposure, confirming high hepatic first-pass effect after oral administration.
Comparison of MCA levels in different tissues of rats that died before their scheduled termination with those sacrificed failed to pinpoint any other target tissue but the kidney as cause of mortality which is compatible with a generalized derailment of energy metabolism. Electrolyte imbalance in the kidney leading to metabolic acidosis may exacerbate the derailment of energy metabolism. Therefore the cause of death of the animals was clearly an overwhelming of the detoxification capacity of the liver and its variability across individual animals. This is at this time the most definitive conclusion because nature in the form of a skin depot provided the ideal experimental conditions of a kinetic steady state during the critical period of intoxication.
High volumes of MCA production, its wide use both in chemical industries as intermediate and in agriculture as postemergence contact herbicide along with very high acute toxicity has resulted in many reported accidental exposures with mostly fatal outcome, both in humans and animals (Kulling et al., 1992; Kusch et al., 1990
; Millischer et al., 1987
; Quick et al., 1983
; Rogers, 1995
). There has been no specific recommended treatment for MCA poisoning that has proven effective. In some cases treatment (e.g., use of antidotes) has actually aggravated toxicity (Bakhisec, 1978
; Chenoweth et al., 1951
; Gosselin et al., 1984
; Hayes et al., 1973
; Kulling et al., 1992
; Kurchatov and Vasileva, 1976
; Kusch et al., 1990
; Millischer et al., 1987
; Rogers, 1995
; Saghir et al., 2001
). For these reasons, it is necessary to discuss the relevance of these findings for accidentally exposed individuals in order to implement effective treatment of patients exposed to potentially lethal doses of MCA.
For oral ingestion, removal of residing chemical from the stomach by gastric lavage should be performed followed by administration of activated charcoal. The danger of aspiration pneumonia may prompt an emergency room physician to forego gastric lavage altogether. In case of dermal exposure, some sort of reverse osmosis needs to be explored that can rapidly and effectively remove sequestered MCA from deeper skin layers. If no method of reverse osmosis can be identified, surgical removal of necrotic skin should be done as the last resort to save the life of the patient followed by reimplanting or grafting of skin after passing of the acute crisis. These procedures should be performed along with the earlier recommended therapies to enhance elimination, improve detoxification capacity, and minimize metabolic acidosis. (Bakhisec, 1978; Kulling et al., 1992
; Kurchatov and Vasileva, 1976
; Kusch et al., 1990
; Rogers, 1995
).
The overall conclusion from three different routes (iv, oral, dermal) of administration of MCA is that it is very difficult, if not impossible, to predict the kinetics of toxic doses from one route to another route of administration. However, without understanding of differential kinetics after different routes of exposure, therapeutic intervention will remain a haphazard undertaking.
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ACKNOWLEDGMENTS |
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NOTES |
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2 To whom correspondence should be addressed at Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd, Kansas City, KS 66160. Fax: (913) 588-7501. E-mail: krozman{at}kumc.edu.
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