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Skin and mucous membrane ulceration in beagle dogs following oral dosing with an experimental aminoglycoside antibiotic
FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
5,986-990
(1985)
Skin and Mucous Membrane Ulceration in Beagle Dogs following Dosing with an Experimental Aminoglycoside Antibiotic PAULT.LAROCCA,FREDBAKER,JERRY D. FRANTZ,ROBERTJ. HUGHE.BLACK, AND EDWARDSCHWARTZ Department
of Toxicology
and Pathologyv
Schering
Corporation,
P.O. Box 32, Lafayette,
Oral
SZOT,
New Jersey
07860
Skin and Mucous Membrane Ulceration in Beagle Dogs following Oral Dosing with an Experimental Aminoglycoside Antibiotic. LA ROCCA, P. T., BAKER,F., FRANTZ,J. D., SZOT, R. J., BLACK,H. E.,ANDSCHWARTZ, E. ( 1985). Fundam. Appl. Toxicol. 5,986-990. G-4 18 is a unique aminoglycoside antibiotic that is structurally related to gentamicin; however, unlike gentamicin, G-4 18 inhibits growth of both procaryotic and eucaryotic cells. In a preliminary acute oral safety study, adult male and female beagles were given a single oral dose of either 2000, 1000, 500,200, or 50 me/kg of G-4 18. Ulceration of the lip, tongue, and/or gingiva occurred in all G-418-dosed dogs 1 to 9 days after dosing. Ulceration of the glans penis, penis sheath, and scrotum occurred 7 to 14 days after a single oral dose with 1000 and 500 mg/kg G-418, and ulceration of the vaginal mucosa of the 200+, lOOO-, 500-, and 50-mg/kg-dosed female dogs occurred 2 to 8 days after dosing. Ulcers of the lip and vaginal area began at the mucocutaneous border and were more severe at these borders. In some dogs a yellow membrane formed over these lesions. Ulceration of the oml and vaginal mucosa disappeared 10 daysafter the first occurrence and reoccurred 3-7 days later. All ulcers healed within 30 days after the single oral dose; however, at necropsy hemorhagic areas of the urinary bladder were observed in at least one of two dogs at each dose level. Similar lesions have not been reported in animals treated with any other aminoglycoside antibiotics. The etiology of these lesions is unknown. o 1985 society of Toxicology.
G-4 18 is a unique aminoglycoside antibiotic that is structurally related to gentamicin (Fig. l), inhibits growth of both procaryotic and eucaryotic cells, and is used extensively in recombinant DNA studies (Davies and Jimenez, 1980; Barclay and Meller, 1983; Kriegler and Botchan, 1983; Van Den Elsen et al., 1983; Ursic et al., 1981). The bactericidal potency of G-418 is less than that of gentamicin and more than that of kanamycin and neomycin (Waitz et al., 1974). In addition to its bactericidal activity, it is also highly active against intestinal protozoa& amoebic, and helminthic infections in rodents (Wagman et al., 1974; Loebenberg et al., 1975). Recently G-4 18 was considered for use as a cestocidal agent in animals because clinical efficacy studies in rodents indicated that it was superior to paromomycin or metronidazole (Loebenberg et al., 1975). An acute oral tox0272-0590/85 $3.00 Copyright 0 1985 by the Society of Toxicolcgy. All rights of reproduction in any form resewed.
986
icity study was conducted to initiate the safety evaluation of this antibiotic. Further studies were not conducted because of adverse reactions observed after single oral doses in dogs. This report describes these reactions and discusses the possible causes. METHODS Purebred vaccinated (distemper, hepatitis, parvo, and rabies) Beagles 10 to 14 months old were used. The dogs were acclimatized to the laboratory for 4 weeks and then randomly assigned to five groups oftwo dogs each (1 male and 1 female) and one group of four dogs (2 males and 2 females). The dogs were fed 300 g of Purina Certified Canine Diet 5007 daily and offered water ad libitum. G-4 18 was given orally in No. 11 gelatin capsules. All dogs were fasted 24 hr prior to dosing. Initially, two male and two female dogs were dosed once with 2000 mgjkg of G-418. Subsequently, single doses of 1000, 500, 200, or 50 mgjkg of G-4 18 were given to one male and one female beagle dog to identify a no-effect level. One group
AMINOGLYCOSIDE
ANTIBIOTIC-INDUCED
tit$ GENTANYCINC
CH-OH
NH2
G-418
FIG. 1. G-4 18 differs from gentamicin only by the addition of hydroxyl groups to the first amino sugar.
of two dogs, given empty gelatin capsules, served as a control. On Day I, each dog was observed immediately, I5 and 30 min, and 1, 3. and 5 hr after dosing. Thereafter each dog was observed twice daily for 14 or 3 1 days. All deviations from the normal physical appearance and behavior were recorded, as were daily food consumption and weekly body weights. Following the observation period, all dogs were sacrificed and complete necropsies were conducted. Only tissueswith gross lesions (tongue, lip, scrotum, penis, vagina, and urinary bladder) were collected and fixed in neutral buffered 10% Formalin and processed for microscopic examination.
RESULTS Emesis, loose stools, lacrimal secretions, interdigital skin irritation, decreased food consumption, and decreased body weights were observed after dosing at all dose levels of G418. A no-effect dose was not found at the levels tested; however, the time-to-appearance of the lesions decreased as the dose increased (dose related). One of two male and one of two female dogs were sacrificed in poor physical condition 8 and 9 days, respectively, after the single oral dose of 2000 mg/kg. These dogs were emaciated, weak, and lethargic. The remaining two
ULCERS
987
2000-mg/kgdosed dogs were necropsied on Day 14. Both of the 2000-mg/kg-dosed male dogs and one of the 2000-mg/kgdosed female dogs had visible blood in their urine from Day 3 to sacrifice. All doses of G-4 18 caused ulceration of the lip, tongue, and/or gingiva 1 to 9 days after dosing (Fig. 2). The lOOO- and 500-mg/kgdosed male dogs had reddening and ulceration of the glans penis, penile sheath, and scrotal area (Fig. 3), which appeared 7 to 14 days after dosing. The 2000-, lOOO-, 500-, and 50-mg/ kg-dosed females had reddening and ulceration of the vaginal mucosa, which appeared 2 to 8 days after dosing. The lip and vaginal lesions began at the mucocutaneous border and were more severe at these borders. In some dogs a yellow membrane formed over these areas. Ulceration of the oral mucosa of all dogs and the vaginal mucosa of all females disappeared 10 days after the first occurrence. All lesions reoccurred spontaneously 3 to 7 days later. All lesions healed within 30 days of the single oral dose. During necropsy of the 2000-mg/kg-dosed dogs hemorrhagic areas of the urinary bladder were observed. Although dogs dosed with 1000 mg/kg or less were necropsied after all external lesions had healed (3 1 days), hemorrhagic areas of the urinary bladder were observed at necropsy in at least one of two dogs at each dose level. No other compound-related gross lesions were observed at necropsy. Microscopic examination of tissues from the G-418-dosed dogs revealed mild to moderate diffuse subacute cystitis with severe diffuse epithelial erosions of the urinary bladder. Focal severe ulceration of the lips and vaginal and penile mucosa occurred at the mucocutaneous junction and was associated with moderate subacute inflammatory infiltration. Moderate to severe subacute inflammatory infiltration was also associated with ulceration of the scrotum. Mild diffise chronic vaginitis occurred in one 2ooO-mg/kgdosed female dog and was considered to be compound related. Since these lesions were unexpected and have not been reported to occur with genta-
988
LA ROCCA ET AL.
FIG. 2. Lesion (with yellow-white membrane over surface) on lip and soft palate appearing 8 days at&x a single oral dose of 50 mg/kg G-4 18.
FIG. 3. Scrotal lesion appearing 7 days after a single oral dose of 500 mg/kg G-418.
AMINOGLYCOSIDE
ANTIBIOTIC-INDUCED
micin or other aminoglycosides, the study was repeated in another group of beagles. The design was similar to that of the previous study with the exception that the ZOOO-mg/kgdose group was eliminated. As in the first study, lesions of the lip, tongue, gingiva, vagina, scrotal area, penile sheath, and glans penis occurred in all dose groups and the time to appearance and severity of the ulcers were the same. The lesions also reoccurred during the same time frame. This study also differed from the first study in that the dogs were allowed to recover for 2 months after dosing. After this 2-month recovery period, they were again dosed with a single oral dose of either 1000, 500,200, or 50 mg/kg G-4 18. Again the results were the same. Lesions of the lip, tongue, gingiva, vagina, scrotal area, penile sheath, and glans penis occurred and the time to appearance and the severity of the lesions were the same as in the two previous studies. DISCUSSION As is characteristic of aminoglycosides, G4 18 is potentially ototoxic and nephrotoxic (Lodhi et al., 1980); however, unlike other aminoglycosides, it inhibits the growth of both procaryotic and eucaryotic cells and, as demonstrated in this report, can cause lesions of the skin and mucous membranes which have not been previously reported for this class of compounds. These lesions are unusual because they are limited to the oral and urogenital areas and they occur after a single dose with no other significant clinical signs of toxicity. The time to appearance of the lesions is dose related and the lesions reoccur without provocation 3 to 7 days after healing. Challenge of previously treated dogs after a 2-month recovery period resulted in similar lesions without a change in the severity or the time to appearance of the lesions. The mechanism by which G-418 caused these lesions could not be determined from these limited studies. The lesions were com-
ULCERS
989
pound related since they did not occur in the control and similar lesions have not been reported in this facility. The specific location of these lesions provides some information about their nature. Local irritation to the oral mucosa as a result of contact with G-4 18 could not have occurred because the compound was given by capsule. In addition, G-4 18 does not appear to be directly irritating to the GI tract (where direct tissue contact occurs) because no GI lesions were observed at necropsy. Aminoglycosides are generally poorly absorbed from the gastrointestinal tract. The data from this study suggest that G-4 18 is absorbed to some extent after oral dosing and we may assume that G4 18 and/or a metabolite which is excreted in the urine and secretions of mucous membranes and salivary glands caused these lesions. The lesions of the genital area could have resulted from licking the genitals and exposure via saliva or by direct contact of urine containing the G-41 8 or metabolite. However, none of these observations can account for the spontaneous reappearance of these lesions several days after healing of the initial lesion, The inability to explain these observations prompt several highly speculative hypotheses. The latency of the lesion, the spontaneous reappearance of the lesions several days after healing, and the similarity of some of the signs to distemper (Hoerlein and Vandevelde, 1978) could suggest that the lesions may be caused by some secondary effect of the drug such as activation of a latent virus. Alternatively, the type of tissue affected (rapidly proliferating cells of skin and mucosa) suggests that the compound may have an effect on rapidly dividing cells. Ulceration of the mucous membranes (mucositis) is a common side effect among antiproliferative drugs (Calabrese and Parks, 1980) and G-4 18 does cause inhibition of both procaryotic and eucaryotic cell growth by inhibiting protein synthesis (Bar-Nun et al., 1983). Cells of the stratum germinativum (stratum basale and stratum spinosum layers) are extremely sensitive to radiation or cytotoxic drugs which act at the DNA/RNA level
990
LA ROCCA ET AL.
(Hobbs and McClellan, 1980; Calabresi and Parks, 1980) and it is not surprising that the first signs of G-4 18 toxicity are lesions involving these cells. Reports of similar lesions in dogs could not be found in the literature. However, there are many reports describing dermatologic syndromes in man which bear some resemblance to the signs observed in dogs dosed with G418. The mechanisms of these reactions in man is believed to involve an immune reaction involving drugs or exogenous antigens from viruses, bacteria, or fungi (Shelly, 1967, 1980; Wuepper et al., 1980; Zugerman and La Voo, 1980). These syndromes include serum sickness syndrome, erythema multiforme, and vesiculitis. These syndromes are dependent not only on the formation of an antibody or immune complex reaction but also on some unique and relatively rare factor that bestows sensitivity to the host. These are rare reactions and, in all probability, would not be manifested in all animals exposed. In contrast, the G-418-induced lesions were observed in all dogs. G-4 I 8 is an interesting aminoglycoside because the lesions are unique within the aminoglycoside family. The etiology of these lesions is unknown; however, interesting parallels exist between the lesions observed in dogs and those observed after exposure to some viruses or bacteria. REFERENCES BARCLAY, S. L., AND MELLER, F. (1983). Efficient transformation of Dictyostelium discoideum amoebae. Mol. Cell Biol. 3, 2 11l-2 130. BAR-NUN, S., SHNEYOUR, Y., AND BECKMANN, J. S. ( 1983). G-4 18, an elongation inhibitor of 80 S ribosomes. B&him. Biophys. Acta 741, 123-127. CA~ABRESI, P., AND PARKS, R. E., JR. (1980). Antiproliferative agents and drugs used for immunosuppression. In The Pharmacological Basis of Therapeutics (A. G. Goodman, L. S. Goodman, and A. Gilman, eds.), pp. 1256-1313. Macmillan Co., New York. DAVIES, J., AND JIMINEZ, A. (1980). A new selective agent
for eukaryotic cloning vectors. Amer. J. Trop. Med. Hyg. 29,1089-1092.
HOBBS,C. H., AND MCCLELLAN, R. 0. (1980). Radiation and radioactive materials. In Toxicology, the Basic Science of Poisons (J. DOUR,C. Klaassen, and M. Amdur, eds.), pp. 497-530. Macmillan Co., New York. HOERLEIN, B. F., AND VANDEVELDE, M. (1978). Primary disorders of the central nervous system.In Canine Neurology (B. F. Hoerlein, ed.), pp. 321-380. Saunders, Philadelphia. KRIEGLER, M., AND BOTCHAN, M. (1983). Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus lons terminal repeat. Mol. Cell Biol. 3, 325-339. LQDHI, S., WEINER, N. D., MECHIGIAN, I., AND SCHACHT, J. (1980). Gtotoxicity of aminoglycosides correlated with their action on monomolecular films of polyphosphoinositides. Biochem. Pharmacol. 29, 597-60 1. LOEBENBERG, D., COUNELIS, M., AND WAITZ, J. A. (1975). Antibiotic G-418, a new micromonospora-produced aminoglycoside with activity against protozoa and helminths: Antiparasitic activity. Antimicrob. Agents Chemother. 7, 8 1l-8 15. SHELLY, W. B. (1967). Herpes simplex virus as a cause of erythema multiforme. J. Amer. Med. Assoc. 201, 7114.
SHELLY, W. B. (1980). Bacterial endotoxin (lipopolysaccharide) as a cause of erythema multiforme. J. Amer. Med. Assoc. 243, 58-60.
URSIC, D., KEMP, J. D., AND HELGESON,J. P. ( 198 1). A new antibiotic with known resistance factors, G-418, inhibits plant cells. Biochem. Biophys. Res. Commun. 101, 1031-1037.
VAN DEN ELSEN, P., HOUWELING, A., AND VAN DER EB, A. (1983). Expression of region Elb of human adenoviruses in the absence of region Ela is not sufficient for complete transformation. Virology 128, 377-390. WAGMAN, G. H., TESTA, R. T., MARQUEZ, J. A., AND WEINSTEIN, M. J. (1974). A new micromonospora-produced aminoglycoside with activity against protozoa and helminths: Determination, isolation, and preliminary characterization. Antimicrob. Agents Chemother. 6, 144149. WAITZ, J. A., SABATELLI, F., MENZEL, F., AND Moss, E. L., JR. ( 1974). Biological activity of antibiotic G-41 8, a new micromonospora-produced aminoglycoside with activity against protozoa and helminths. Antimicrob. Agents Chemother. 6, 579-58 1. WUEPPER, K. D., WATSON, P. A., AND KAZMIEROWSKI, J. A. (1980). Immune complexes in erythema multiforme and Stevers-Johnson syndrome. J. Invest. Dermatol. 74, 368-37 1. ZUGERMAN, C., AND LA Voo, E. J. (1980). Erythema multiforme caused by oral Furosemide. Arch. Dermatol. 116,518-519.
AND
APPLIED
TOXICOLOGY
5,986-990
(1985)
Skin and Mucous Membrane Ulceration in Beagle Dogs following Dosing with an Experimental Aminoglycoside Antibiotic PAULT.LAROCCA,FREDBAKER,JERRY D. FRANTZ,ROBERTJ. HUGHE.BLACK, AND EDWARDSCHWARTZ Department
of Toxicology
and Pathologyv
Schering
Corporation,
P.O. Box 32, Lafayette,
Oral
SZOT,
New Jersey
07860
Skin and Mucous Membrane Ulceration in Beagle Dogs following Oral Dosing with an Experimental Aminoglycoside Antibiotic. LA ROCCA, P. T., BAKER,F., FRANTZ,J. D., SZOT, R. J., BLACK,H. E.,ANDSCHWARTZ, E. ( 1985). Fundam. Appl. Toxicol. 5,986-990. G-4 18 is a unique aminoglycoside antibiotic that is structurally related to gentamicin; however, unlike gentamicin, G-4 18 inhibits growth of both procaryotic and eucaryotic cells. In a preliminary acute oral safety study, adult male and female beagles were given a single oral dose of either 2000, 1000, 500,200, or 50 me/kg of G-4 18. Ulceration of the lip, tongue, and/or gingiva occurred in all G-418-dosed dogs 1 to 9 days after dosing. Ulceration of the glans penis, penis sheath, and scrotum occurred 7 to 14 days after a single oral dose with 1000 and 500 mg/kg G-418, and ulceration of the vaginal mucosa of the 200+, lOOO-, 500-, and 50-mg/kg-dosed female dogs occurred 2 to 8 days after dosing. Ulcers of the lip and vaginal area began at the mucocutaneous border and were more severe at these borders. In some dogs a yellow membrane formed over these lesions. Ulceration of the oml and vaginal mucosa disappeared 10 daysafter the first occurrence and reoccurred 3-7 days later. All ulcers healed within 30 days after the single oral dose; however, at necropsy hemorhagic areas of the urinary bladder were observed in at least one of two dogs at each dose level. Similar lesions have not been reported in animals treated with any other aminoglycoside antibiotics. The etiology of these lesions is unknown. o 1985 society of Toxicology.
G-4 18 is a unique aminoglycoside antibiotic that is structurally related to gentamicin (Fig. l), inhibits growth of both procaryotic and eucaryotic cells, and is used extensively in recombinant DNA studies (Davies and Jimenez, 1980; Barclay and Meller, 1983; Kriegler and Botchan, 1983; Van Den Elsen et al., 1983; Ursic et al., 1981). The bactericidal potency of G-418 is less than that of gentamicin and more than that of kanamycin and neomycin (Waitz et al., 1974). In addition to its bactericidal activity, it is also highly active against intestinal protozoa& amoebic, and helminthic infections in rodents (Wagman et al., 1974; Loebenberg et al., 1975). Recently G-4 18 was considered for use as a cestocidal agent in animals because clinical efficacy studies in rodents indicated that it was superior to paromomycin or metronidazole (Loebenberg et al., 1975). An acute oral tox0272-0590/85 $3.00 Copyright 0 1985 by the Society of Toxicolcgy. All rights of reproduction in any form resewed.
986
icity study was conducted to initiate the safety evaluation of this antibiotic. Further studies were not conducted because of adverse reactions observed after single oral doses in dogs. This report describes these reactions and discusses the possible causes. METHODS Purebred vaccinated (distemper, hepatitis, parvo, and rabies) Beagles 10 to 14 months old were used. The dogs were acclimatized to the laboratory for 4 weeks and then randomly assigned to five groups oftwo dogs each (1 male and 1 female) and one group of four dogs (2 males and 2 females). The dogs were fed 300 g of Purina Certified Canine Diet 5007 daily and offered water ad libitum. G-4 18 was given orally in No. 11 gelatin capsules. All dogs were fasted 24 hr prior to dosing. Initially, two male and two female dogs were dosed once with 2000 mgjkg of G-418. Subsequently, single doses of 1000, 500, 200, or 50 mgjkg of G-4 18 were given to one male and one female beagle dog to identify a no-effect level. One group
AMINOGLYCOSIDE
ANTIBIOTIC-INDUCED
tit$ GENTANYCINC
CH-OH
NH2
G-418
FIG. 1. G-4 18 differs from gentamicin only by the addition of hydroxyl groups to the first amino sugar.
of two dogs, given empty gelatin capsules, served as a control. On Day I, each dog was observed immediately, I5 and 30 min, and 1, 3. and 5 hr after dosing. Thereafter each dog was observed twice daily for 14 or 3 1 days. All deviations from the normal physical appearance and behavior were recorded, as were daily food consumption and weekly body weights. Following the observation period, all dogs were sacrificed and complete necropsies were conducted. Only tissueswith gross lesions (tongue, lip, scrotum, penis, vagina, and urinary bladder) were collected and fixed in neutral buffered 10% Formalin and processed for microscopic examination.
RESULTS Emesis, loose stools, lacrimal secretions, interdigital skin irritation, decreased food consumption, and decreased body weights were observed after dosing at all dose levels of G418. A no-effect dose was not found at the levels tested; however, the time-to-appearance of the lesions decreased as the dose increased (dose related). One of two male and one of two female dogs were sacrificed in poor physical condition 8 and 9 days, respectively, after the single oral dose of 2000 mg/kg. These dogs were emaciated, weak, and lethargic. The remaining two
ULCERS
987
2000-mg/kgdosed dogs were necropsied on Day 14. Both of the 2000-mg/kg-dosed male dogs and one of the 2000-mg/kgdosed female dogs had visible blood in their urine from Day 3 to sacrifice. All doses of G-4 18 caused ulceration of the lip, tongue, and/or gingiva 1 to 9 days after dosing (Fig. 2). The lOOO- and 500-mg/kgdosed male dogs had reddening and ulceration of the glans penis, penile sheath, and scrotal area (Fig. 3), which appeared 7 to 14 days after dosing. The 2000-, lOOO-, 500-, and 50-mg/ kg-dosed females had reddening and ulceration of the vaginal mucosa, which appeared 2 to 8 days after dosing. The lip and vaginal lesions began at the mucocutaneous border and were more severe at these borders. In some dogs a yellow membrane formed over these areas. Ulceration of the oral mucosa of all dogs and the vaginal mucosa of all females disappeared 10 days after the first occurrence. All lesions reoccurred spontaneously 3 to 7 days later. All lesions healed within 30 days of the single oral dose. During necropsy of the 2000-mg/kg-dosed dogs hemorrhagic areas of the urinary bladder were observed. Although dogs dosed with 1000 mg/kg or less were necropsied after all external lesions had healed (3 1 days), hemorrhagic areas of the urinary bladder were observed at necropsy in at least one of two dogs at each dose level. No other compound-related gross lesions were observed at necropsy. Microscopic examination of tissues from the G-418-dosed dogs revealed mild to moderate diffuse subacute cystitis with severe diffuse epithelial erosions of the urinary bladder. Focal severe ulceration of the lips and vaginal and penile mucosa occurred at the mucocutaneous junction and was associated with moderate subacute inflammatory infiltration. Moderate to severe subacute inflammatory infiltration was also associated with ulceration of the scrotum. Mild diffise chronic vaginitis occurred in one 2ooO-mg/kgdosed female dog and was considered to be compound related. Since these lesions were unexpected and have not been reported to occur with genta-
988
LA ROCCA ET AL.
FIG. 2. Lesion (with yellow-white membrane over surface) on lip and soft palate appearing 8 days at&x a single oral dose of 50 mg/kg G-4 18.
FIG. 3. Scrotal lesion appearing 7 days after a single oral dose of 500 mg/kg G-418.
AMINOGLYCOSIDE
ANTIBIOTIC-INDUCED
micin or other aminoglycosides, the study was repeated in another group of beagles. The design was similar to that of the previous study with the exception that the ZOOO-mg/kgdose group was eliminated. As in the first study, lesions of the lip, tongue, gingiva, vagina, scrotal area, penile sheath, and glans penis occurred in all dose groups and the time to appearance and severity of the ulcers were the same. The lesions also reoccurred during the same time frame. This study also differed from the first study in that the dogs were allowed to recover for 2 months after dosing. After this 2-month recovery period, they were again dosed with a single oral dose of either 1000, 500,200, or 50 mg/kg G-4 18. Again the results were the same. Lesions of the lip, tongue, gingiva, vagina, scrotal area, penile sheath, and glans penis occurred and the time to appearance and the severity of the lesions were the same as in the two previous studies. DISCUSSION As is characteristic of aminoglycosides, G4 18 is potentially ototoxic and nephrotoxic (Lodhi et al., 1980); however, unlike other aminoglycosides, it inhibits the growth of both procaryotic and eucaryotic cells and, as demonstrated in this report, can cause lesions of the skin and mucous membranes which have not been previously reported for this class of compounds. These lesions are unusual because they are limited to the oral and urogenital areas and they occur after a single dose with no other significant clinical signs of toxicity. The time to appearance of the lesions is dose related and the lesions reoccur without provocation 3 to 7 days after healing. Challenge of previously treated dogs after a 2-month recovery period resulted in similar lesions without a change in the severity or the time to appearance of the lesions. The mechanism by which G-418 caused these lesions could not be determined from these limited studies. The lesions were com-
ULCERS
989
pound related since they did not occur in the control and similar lesions have not been reported in this facility. The specific location of these lesions provides some information about their nature. Local irritation to the oral mucosa as a result of contact with G-4 18 could not have occurred because the compound was given by capsule. In addition, G-4 18 does not appear to be directly irritating to the GI tract (where direct tissue contact occurs) because no GI lesions were observed at necropsy. Aminoglycosides are generally poorly absorbed from the gastrointestinal tract. The data from this study suggest that G-4 18 is absorbed to some extent after oral dosing and we may assume that G4 18 and/or a metabolite which is excreted in the urine and secretions of mucous membranes and salivary glands caused these lesions. The lesions of the genital area could have resulted from licking the genitals and exposure via saliva or by direct contact of urine containing the G-41 8 or metabolite. However, none of these observations can account for the spontaneous reappearance of these lesions several days after healing of the initial lesion, The inability to explain these observations prompt several highly speculative hypotheses. The latency of the lesion, the spontaneous reappearance of the lesions several days after healing, and the similarity of some of the signs to distemper (Hoerlein and Vandevelde, 1978) could suggest that the lesions may be caused by some secondary effect of the drug such as activation of a latent virus. Alternatively, the type of tissue affected (rapidly proliferating cells of skin and mucosa) suggests that the compound may have an effect on rapidly dividing cells. Ulceration of the mucous membranes (mucositis) is a common side effect among antiproliferative drugs (Calabrese and Parks, 1980) and G-4 18 does cause inhibition of both procaryotic and eucaryotic cell growth by inhibiting protein synthesis (Bar-Nun et al., 1983). Cells of the stratum germinativum (stratum basale and stratum spinosum layers) are extremely sensitive to radiation or cytotoxic drugs which act at the DNA/RNA level
990
LA ROCCA ET AL.
(Hobbs and McClellan, 1980; Calabresi and Parks, 1980) and it is not surprising that the first signs of G-4 18 toxicity are lesions involving these cells. Reports of similar lesions in dogs could not be found in the literature. However, there are many reports describing dermatologic syndromes in man which bear some resemblance to the signs observed in dogs dosed with G418. The mechanisms of these reactions in man is believed to involve an immune reaction involving drugs or exogenous antigens from viruses, bacteria, or fungi (Shelly, 1967, 1980; Wuepper et al., 1980; Zugerman and La Voo, 1980). These syndromes include serum sickness syndrome, erythema multiforme, and vesiculitis. These syndromes are dependent not only on the formation of an antibody or immune complex reaction but also on some unique and relatively rare factor that bestows sensitivity to the host. These are rare reactions and, in all probability, would not be manifested in all animals exposed. In contrast, the G-418-induced lesions were observed in all dogs. G-4 I 8 is an interesting aminoglycoside because the lesions are unique within the aminoglycoside family. The etiology of these lesions is unknown; however, interesting parallels exist between the lesions observed in dogs and those observed after exposure to some viruses or bacteria. REFERENCES BARCLAY, S. L., AND MELLER, F. (1983). Efficient transformation of Dictyostelium discoideum amoebae. Mol. Cell Biol. 3, 2 11l-2 130. BAR-NUN, S., SHNEYOUR, Y., AND BECKMANN, J. S. ( 1983). G-4 18, an elongation inhibitor of 80 S ribosomes. B&him. Biophys. Acta 741, 123-127. CA~ABRESI, P., AND PARKS, R. E., JR. (1980). Antiproliferative agents and drugs used for immunosuppression. In The Pharmacological Basis of Therapeutics (A. G. Goodman, L. S. Goodman, and A. Gilman, eds.), pp. 1256-1313. Macmillan Co., New York. DAVIES, J., AND JIMINEZ, A. (1980). A new selective agent
for eukaryotic cloning vectors. Amer. J. Trop. Med. Hyg. 29,1089-1092.
HOBBS,C. H., AND MCCLELLAN, R. 0. (1980). Radiation and radioactive materials. In Toxicology, the Basic Science of Poisons (J. DOUR,C. Klaassen, and M. Amdur, eds.), pp. 497-530. Macmillan Co., New York. HOERLEIN, B. F., AND VANDEVELDE, M. (1978). Primary disorders of the central nervous system.In Canine Neurology (B. F. Hoerlein, ed.), pp. 321-380. Saunders, Philadelphia. KRIEGLER, M., AND BOTCHAN, M. (1983). Enhanced transformation by a simian virus 40 recombinant virus containing a Harvey murine sarcoma virus lons terminal repeat. Mol. Cell Biol. 3, 325-339. LQDHI, S., WEINER, N. D., MECHIGIAN, I., AND SCHACHT, J. (1980). Gtotoxicity of aminoglycosides correlated with their action on monomolecular films of polyphosphoinositides. Biochem. Pharmacol. 29, 597-60 1. LOEBENBERG, D., COUNELIS, M., AND WAITZ, J. A. (1975). Antibiotic G-418, a new micromonospora-produced aminoglycoside with activity against protozoa and helminths: Antiparasitic activity. Antimicrob. Agents Chemother. 7, 8 1l-8 15. SHELLY, W. B. (1967). Herpes simplex virus as a cause of erythema multiforme. J. Amer. Med. Assoc. 201, 7114.
SHELLY, W. B. (1980). Bacterial endotoxin (lipopolysaccharide) as a cause of erythema multiforme. J. Amer. Med. Assoc. 243, 58-60.
URSIC, D., KEMP, J. D., AND HELGESON,J. P. ( 198 1). A new antibiotic with known resistance factors, G-418, inhibits plant cells. Biochem. Biophys. Res. Commun. 101, 1031-1037.
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