Hematological Effects in F344 Rats and B6C3F1 Mice during the 13Week Gavage Toxicity Study of Methylene Blue Trihydrate
M. R. Hejtmancik*,1,
M. J. Ryan*,
J. D. Toft*,
R. L. Persing*,
P. J. Kurtz* and
R. S. Chhabra
* Battelle, 505 King Avenue, Columbus, Ohio 43201; and
Division of Toxicology Research and Testing Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709
Received June 13, 2001;
accepted September 28, 2001
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ABSTRACT
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Methylene blue trihydrate is used widely as a dye and therapeutic agent. Methylene blue was administered by gavage to 30 animals/sex/dose group in a 0.5% aqueous methylcellulose suspension at doses of 0, 25, 50, 100, and 200 mg/kg. Blood samples from 10 animals/sex/dose group were collected at the end of study weeks 1, 6, and 13. Methylene blue treatment resulted in methemoglobin formation and oxidative damage to red blood cells, leading to a regenerative anemia and a variety of tissue and biochemical changes secondary to erythrocyte injury. An early change was a dose-related increase in methemoglobin, where the response of rats and mice was similar in magnitude. Mice appeared to be more sensitive than rats to the formation of Heinz bodies and the development of anemia that was characterized by a decrease in hemoglobin, hematocrit, and erythrocyte count. Splenomegaly was apparent in all treated mice and in the 100 mg/kg (males only) and 200 mg/kg rats at necropsy. There was a dose-related increase in absolute and relative spleen weight for both species. Microscopic examination revealed increased splenic hematopoiesis in all mice treatment groups and in rats at the 50 mg/kg dose level and above. Splenic congestion and bone marrow hyperplasia were also observed in these rat-dose groups. Mice at the higher doses showed hematopoiesis in the liver and accumulation of hemosiderin in Kupffer cells. These gross and microscopic findings are consistent with the development of hemolytic anemia. A dose-related increase in the reticulocyte count during study weeks 6 and 13 suggested a compensatory response to anemia.
Key Words: methylene blue trihydrate; F344 rats; B6C3F1 mice; methemoglobin and Heinz body formation; splenomegaly; hematopoiesis.
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INTRODUCTION
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Methylene blue trihydrate is used therapeutically in the treatment of acquired and idiopathic methemoglobinemia. In low concentrations, methylene blue increases the conversion of methemoglobin to hemoglobin. Oral administration of 100 to 300 mg/day of methylene blue causes a marked reduction in methemoglobin levels in humans with methemoglobin reductase deficiency (Bunn, 1994
). Severe toxic methemoglobinemia is treated by the iv administration of methylene blue (2 mg/kg), which can reduce the methemoglobin concentration by at least 50% within 1 h. Methylene blue is converted by reductases in erythrocytes to leukomethylene blue, which reduces methylene blue to hemoglobin. In higher concentrations, methylene blue oxidizes ferrous iron in hemoglobin to the ferric state, producing methemoglobin. In the treatment of cyanide, methemoglobin combines with cyanide to produce cyanomethemoglobin, preventing the interference of cyanide with the cytochrome system. While methylene blue was previously used to produce methemoglobinemia in the treatment of cyanide poisoning, sodium nitrite is considered to be a more effective and safer alternative. Other medicinal uses of methylene blue include the treatment of chronic urolithiasis, cutaneous viral infections, and manic-depressive psychosis (HSDB, 1999
). As a dye/stain, methylene blue is used in surgical and medical marking and as an indicator dye, a bacteriological stain, a food colorant, and as a dye for cotton and wool. In spite of the medicinal/therapeutic use of methylene blue and the potential for human exposure, a review by the National Cancer Institute revealed a paucity of information concerning prechronic and chronic toxicity (NTP, 1990
). In veterinary medicine, methylene blue has been prohibited to treat methemoglobinemia in animals raised to produce meat for human consumption. A recommendation for further testing was predicated on the inadequate toxicity database in animals, a high potential for human and animal exposure, numerous short-term studies with inconsistent results, and the lack of long-term toxicity data, including epidemiological studies. A 14-day repeated-dose gavage study (Trela et al., report submitted to NTP, 1992) was performed to provide information to determine the doses for the 13-week study reported here. In the 14-day study, methylene blue-induced toxicity included a hypoxic hemolytic anemia due to the formation of methemoglobin and Heinz bodies that resulted in the early deaths of rats at doses of 500 mg/kg and above and of mice at doses of 250 mg/kg and above.
Hematological abnormalities have been described in several species following the administration of methylene blue (Perry and Meinhard, 1974
). One study examined and compared the responses of several species to methylene blue. A single ip injection of 20 to 100 mg/kg was administered to cats, dogs, and guinea pigs and from 20 to 200 mg/kg to mice, rabbits, and rats (Rentsch and Wittekind, 1967
). The tolerance for methylene blue varied considerably in the different animal species. Within several days following treatment, most species showed a decrease in erythrocytes and hemoglobin with an increase in reticulocytes. The biggest difference among species was the capability to form Heinz bodies. Cats and dogs were the most sensitive species tested with Heinz bodies detectable 4 and 6 h, respectively, after methylene blue administration. At 200 mg/kg, Heinz bodies were detected in all treated mice within 24 h after administration. Rats were found to be less sensitive than mice. At 200 mg/kg, only 12% of the treated rats showed the formation of Heinz bodies within 96 h of treatment. At 200 mg/kg, 70% of treated rabbits developed Heinz bodies within 96 h of treatment. Guinea pigs were the most resistant with only 4% of treated animals (100 mg/kg) showing Heinz bodies within a 72-h period. Cats given a single po dose of methylene blue (10 to 20 mg/kg) showed only a small increase in methemoglobin but a large increase in the formation of Heinz bodies (Spicer and Thompson, 1949
). The authors attributed the ability to produce Heinz bodies of various oxidizing agents to the duration of action. Nitrite, a short-acting substance, yields high levels of methemoglobin without Heinz bodies. Methylene blue, with a much longer duration of action, produces high levels of Heinz bodies with little or no methemoglobin. Sprague-Dawley rats (Stossel and Jennings, 1966
) given a single ip injection of methylene at the approximate LD50 level (65 mg/kg) and mongrel dogs (Stossel, 1968a
,b
) receiving up to 35 mg/kg methylene blue by iv infusion showed no increases in blood methemoglobin concentrations. Due to the variety of hematological effects observed in several species, extra animals were added to the experimental design of these subchronic studies for the periodic assessment of hematological effects.
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MATERIALS AND METHODS
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Chemicals.
Methylene blue trihydrate (CAS No. 61-73-4) was obtained in a single lot from Aldrich Chemical Company (Milwaukee, WI). A chemical reanalysis revealed a purity of 98.5% based upon analysis by high performance liquid chromatography (HPLC). Periodic bulk chemical analyses by HPLC revealed comparable purity results and indicated no degradation during storage at room temperature (
25°C) protected from light. Doses were formulated for gavage as a suspension in 0.5% aqueous methylcellulose. The dosage concentrations were 0, 5, 10, 20, and 40 mg/ml for rats and 0, 2.5, 5, 10, and 20 mg/ml for mice. Prior to the study, a 35-day stability was established for the 2.5 mg/ml suspension, which was the lowest concentration administered. Homogeneity of the highest (40 mg/ml) concentration of suspension was demonstrated prior to the study. Gavage dosing suspensions were prepared once every 4 weeks, placed in glass bottles, and were stored at room temperature until used for dosing. A different dosing bottle was prepared for each dosing day. A stir bar was used prior to dosing to ensure homogeneity of the suspension. All preadministration dose analyses indicated concentrations were within 10% of the target concentration. Mid-study postadministration dose analyses agreed with preadministration values, indicating no decomposition or degradation occurred during animal treatment.
Animals.
All animals were obtained from Taconic Laboratory Animals and Services (Germantown, NY). F344 rats and B6C3F1 mice were shipped at approximately 4 weeks of age, were quarantined for 12 days, and were approximately 6 weeks of age at the start of the study. Animals were randomly assigned to dose groups by sex and body weight by partitioning algorithm using a XybionTM computer program (Cedar Knolls, NJ). There were no statistically significant differences between group mean body weights prior to the initiation of these studies. All rats and female mice were housed 5/cage, and male mice were individually housed in polycarbonate cages with hardwood chips as bedding. NIH-07 feed pellets (Zeigler Brothers, Inc., Gardners, PA) and tap water were available ad libitum. The animal room temperature was approximately 72 ± 3°F and humidity was 50 ± 15%. Fluorescent lights were on for 12 h/day and there were a minimum of 10 room air changes per h.
Experimental design.
Groups of 10 animals of each sex and species (core study) were given methylene blue trihydrate by gavage at doses of 0, 25, 50, 100, and 200 mg/kg body weight, once a day, 5 days/week, for 13 weeks. This study was performed in accordance with the Specifications for the Conduct of NIEHS Studies, which specifies dosing on weekdays only. The volume of vehicle and dose suspension administered was 5 ml/kg for rats and 10 ml/kg for mice. Extra animals (10/sex/dose group/time period) were included for clinical pathology studies that were performed on Study Days 8 and 43. Blood samples were obtained from core study animals at study termination (Day 92). Animals were anesthetized with a mixture of carbon dioxide and oxygen prior to bleeding, and blood was collected by retro-orbital plexus puncture (rats) or cardiac puncture (mice). Animals always received at least 2 consecutive dose administrations, with the last dose administered approximately 30 min prior to bleeding. Blood was collected into micro-collection tubes (Sarstedt, Inc., Newton, NC) containing potassium-EDTA for hematology studies and into serum separator tubes to obtain samples for clinical chemistry. Hematological analyses were performed with a Serono-Baker System 9000 Hematology Analyzer (Allentown, PA). Blood methemoglobin concentrations were determined by the spectrophotometric method of Evelyn and Malloy (1938). Serum chemistry determinations were performed using a Hitachi 704 Chemistry Analyzer (Indianapolis, IN) and included blood urea nitrogen; serum creatinine, bile salts, total protein, and albumin; and the activities of alanine aminotransferase (SGPT), alkaline phosphatase, sorbitol dehydrogenase, and creatine kinase.
Animals were checked twice daily for moribundity or morbidity, and were examined once weekly for clinical signs of toxicity. The individual animal body weights were recorded once weekly, and the most recent individual body weight was used to determine dosing volume. At study termination, animals were bled for clinical pathology studies approximately 30 min following the last dose administration just prior to necropsy. Animals were weighed and euthanized with carbon dioxide.
Necropsy was performed on all core study animals, including early deaths. At study termination, selected organ weights (liver, thymus, right kidney, right testis, heart, spleen, and lungs) were taken. All collected tissues were preserved in 10% neutral buffered formalin. Tissues examined microscopically in all control, high dose (200 mg/kg), and early death animals included adrenal glands, brain, clitoral glands, esophagus, bone marrow (femur), gallbladder (mice), heart (aorta), large intestine (cecum, colon, rectum), small intestine (duodenum, jejunum, ileum), kidneys, liver, lungs including mainstem bronchi, lymph nodes (mandibular, mesenteric), mammary gland, nasal cavity (including turbinates), ovaries, pancreas, parathyroid glands, pituitary gland, preputial gland, prostate, salivary glands, seminal vesicles, spleen, stomach (forestomach and glandular), testis with epididymis, thymus, thyroid gland, trachea, urinary bladder, uterus, and any gross lesions seen at necropsy. Target organs that were examined in animals in the lower dose groups included the spleen and bone marrow in rats and the spleen and liver in mice. The severity of lesions was graded from minimal to marked on a 14 scale.
In-life data (body weights, clinical observations) and microscopic findings were collected and summarized using a computerized system (Toxicology Data Management System) provided by the National Toxicology Program (NTP, 1998
). Body weight data were compared using Fisher's least significant difference method. Organ weight and clinical pathology data were summarized and analyzed statistically using the XybionTM Pathology/Toxicology computer system. Organ weight data and clinical pathology data were tested for homogeneity of variance by Bartlett's test. For data that were not homogenous, Dunnett's test was performed. For homogenous data, a 1-way analysis of variance (ANOVA) was performed, followed by Dunnett's test.
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RESULTS
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The survival and group mean body weights at study termination are included in Table 1
for rats and mice. The causes of death of 6 rats and 2 mice that were found dead over the course of this study were attributed to gavage error and substantiated by necropsy findings in the lungs (discoloration), thoracic cavity (fluid), and esophagus or trachea (perforation). This abnormally high gavage accident rate in treated animals was attributed to the technical difficulty of administering a heavily pigmented and viscous solution. Survival (60% or greater) was adequate in treated groups to assess toxicological effects. At study termination, high dose (200 mg/kg) male rats and male mice showed statistically significant reductions in body weight relative to control of approximately 6 and 10%, respectively.
Erythrocyte counts and hematocrit values for rats are shown in Table 2
. Hemoglobin concentrations for rats are included in Table 3
. There were no treatment-related changes in these parameters after 1 week of treatment, except for a decreased hemoglobin concentration in high dose female rats. After 6 weeks of treatment, male and female rats showed a dose-related decrease in erythrocyte count and treated male rats showed a decrease in hematocrit. Male and female rats showed a decrease in the concentration of hemoglobin and the mean corpuscular hemoglobin concentration (high dose females only). Hematocrit values during Week 13 were significantly different in high dose male rats and females at 50 mg/kg and above. At 13 weeks, male and female rats both showed dose-related reductions in erythrocyte counts relative to control. The hematocrit and hemoglobin concentration was reduced in the 200 mg/kg male group and the 50, 100, and 200 mg/kg female group, but the mean corpuscular hemoglobin concentration was similar to control.
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TABLE 2 Hematocrit and RBC Counts during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in F344 Rats
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TABLE 3 Hemoglobin and Mean Corpuscular Hemoglobin Concentration during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in F344 Rats
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Erythrocyte counts and hematocrit values for mice are shown in Table 4
. Mice showed dose-related reductions in hematocrit values and erythrocyte counts that were present after 1, 6, and 13 weeks of treatment. In general, the greatest magnitude of change appeared to occur during Week 6 for male mice and during Week 13 for female mice. At similar doses, responses in the mice were generally greater than those of rats. Mice also showed a decrease in hemoglobin concentration but an increase in mean corpuscular hemoglobin concentration (Table 5
). In both species, treatment caused a compensatory dose-related increase in reticulocytes and mean corpuscular volume (Table 6
for rats and Table 7
for mice). At similar doses, the reticulocyte response in mice surpassed that of rats.
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TABLE 4 Hematocrit and RBC Counts during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in B6C3F1 Mice
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TABLE 5 Hemoglobin and Mean Corpuscular Hemoglobin Concentration during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in B6C3F1 Mice
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TABLE 6 Reticulocyte Counts and Mean Corpuscular Volume during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in F344 Rats
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TABLE 7 Reticulocyte Counts and Mean Corpuscular Volume during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in B6C3F1 Mice
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Blood methemoglobin concentrations for rats and mice are shown in Table 8
. At each time period, a small treatment-related increase occurred in blood methemoglobin concentrations that was generally dose-related. At similar dose levels, the responses of the rat and mice treatment groups were similar in magnitude.
The percentage of erythrocytes containing Heinz bodies is shown in Table 9
for rats and mice. In rats, an increase in Heinz bodies occurred predominately in high dose animals during the latter phase (Weeks 6 and 13) of the study. In general, mice showed more pronounced increases and these occurred earlier in the study (Week 1) and persisted until study termination. Exposure to methylene blue produced no consistent or treatment-related changes in any of the serum chemistry parameters that were evaluated in rats or mice (data not shown).
Absolute spleen weights and spleen-body weight ratios for rats and mice are included in Table 10
. Treatment was associated with a dose-related increase in both absolute and relative spleen weight. Splenomegaly was observed in all methylene blue mice treatment groups at study termination in a dose-response pattern.
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TABLE 10 Summary of Spleen Weights and Spleen-Weight-to-Body-Weight Ratios during the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate
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Microscopic findings in rats are summarized in Table 11
. Microscopic examination revealed abnormalities in the 50 mg/kg and above male and female dose groups, including hyperplasia of the bone marrow with splenic congestion and hematopoietic cell proliferation of the spleen. Depletion of cellular elements in splenic lymphoid follicles occurred in male and female rats in the 100 and 200 mg/kg dose groups.
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TABLE 11 Incidence of Microscopic Lesions following the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in F344 Rats
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Microscopic findings in mice are summarized in Table 12
. Microscopic examination revealed splenic hematopoiesis in all treatment groups. At similar doses, the extent of hematopoiesis was generally more severe in mice than rats. Mice at the higher dose levels showed hematopoiesis of the liver and accumulation of hemosiderin in Kupffer cells.
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TABLE 12 Incidence of Microscopic Changes following the 13-Week Gavage Toxicity Study of Methylene Blue Trihydrate in B6C3F1 Mice
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DISCUSSION
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Methylene blue produced toxicity of the hematopoietic system. Methemoglobinemia was the primary toxic response. Other changes occurred secondary to methemoglobin formation and included subsequent increases in erythrocyte injury and turnover. These abnormalities include red blood cell morphological alterations (e.g., Heinz bodies) and changes indicative of anemia. Methylene blue produced a compensatory anemia as evidenced by an increase in the reticulocyte count and a decrease in hemoglobin concentrations, hematocrit, and red blood cell counts. The anemia was macrocytic, as indicated by an increase in mean cell volume in rats and mice, and hyperchromic, as indicated by an increase in mean cell hemoglobin concentration (mice only). The macrocytosis was attributed to an increased number of larger reticulocytes in circulation. The hyperchromia indicated increased red blood cell fragility and the release of hemoglobin in the plasma. The presence of Heinz bodies was consistent with direct oxidative damage to the red cell by methylene blue (Harvey, 1989
). Increased hematopoietic cell proliferation is attributed to erythrocyte injury and increased turnover.
Early changes that occurred after one week of treatment included the production of methemoglobin (rats and mice) and the appearance of Heinz bodies (mice). Other hematological parameters (hematocrit, hemoglobin, erythrocyte count) were normal in rats but were decreased in mice. Assessment of hematology parameters during subsequent study weeks revealed that many of these changes were progressive with continued treatment. Hematological parameters in rats and mice showed changes indicative of hemolytic anemia at study weeks 6 and 13. The decrease in hematocrit and mean corpuscular hemoglobin concentration that occurred in all male rat dosage groups at 6 weeks did not occur at 13 weeks, except for the decrease in hematocrit of the high dosage (200 mg/kg) male rats. These effects in male rats subsided, indicating some recovery with continued treatment. These changes are consistent with bone marrow hyperplasia and hematopoietic cell proliferation that were observed in treated male rats at study termination.
In this study, a dose-related increase in spleen weight (absolute and spleen-to-body-weight ratio) and in spleen size was apparent at study termination in both species. Splenomegaly was attributed to the sequestration of damaged erythrocytes in splenic sinusoids and increased hematopoietic elements. These splenic effects appeared to be secondary to erythrocyte toxicity, since there were no direct or primary microscopic abnormalities observed in the spleen. The development of hemolytic anemia produced a compensatory erythropoiesis, which was observed in the spleen and liver (mice only) and associated with an increased production of reticulocytes.
The development of toxicity was similar in nature to that produced by treatment with other methemoglobin producers such as aniline (Gralla et al., 1979
) and its chlorinated derivatives (Chhabra et al., 1991
; Hejtmancik et al., manuscript in preparation). Although they were statistically significant, methylene blue caused increases in methemoglobin that were relatively small in magnitude (
1 g/dl). Treatment of rats and mice with 10 to 160 mg/kg ortho- or meta-chloroaniline was shown to increase methemoglobin to concentrations as high as approximately 5 g/dl in mice and 7 g/dl in mice (NTP, 1998
). A diminution in the methemoglobin response in animals exposed to methylene blue may have resulted in part from stimulation of the dormant NADPH-reductase system. At low doses (2 mg/kg iv), methylene blue produces stimulation of this reductase system and is administered therapeutically to remedy methemoglobinemia. Paradoxically higher doses cause oxidation of hemoglobin to methemoglobin. The generally lower levels of methemoglobin in rats compared to mice given similar methylene blue doses are consistent with reports that rats have greater reductase capacity than mice (Smith, 1996
). An increase in reductase activity of rats compared to mice may have retarded Heinz body formation, since both effects result from the oxidation of a substrate by methylene blue.
These subchronic studies in rats and mice suggest that subchronic exposure to methylene blue causes hematological abnormalities that are progressive in development. Several methemoglobin-producing chemicals have been shown to induce splenic nonlymphoid sarcomas. Examples are aniline (Goodman et al., 1984
) and a number of structurally related aniline compounds (Chhabra et al., 1991
; Goodman et al., 1984
). Other than lymphomas, tumors of the spleen are highly uncommon in laboratory rats and mice (Stefanski et al., 1990
). A mechanism has been proposed for the induction of rare spleen tumors by chemicals that produce methemoglobin (Goodman et al., 1984
). Repeated exposure might cause a sustained stimulation of the hematopoietic system. Persistent stimulation of the spleen may produce fibrosis, a preneoplastic lesion that could progress to sarcoma. If methemoglobin formation is used as a predictor of this nongenetic mechanism for induction of spleen tumors, methylene blue should be considered for chronic study evaluations.
These subchronic studies were performed to identify any differences in sensitivity between species and sexes to methylene blue and to provide information for dosage selection for chronic carcinogenicity studies. The no observed adverse effect level (NOAEL) in rats was 25 mg/kg due to the absence of splenic congestion and hematopoietic cell proliferation that occurred at the higher dosage levels (50 mg/kg and above). The NOAEL in mice was also determined to be 25 mg/kg due to the low incidence and minor severity of microscopic changes.
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NOTES
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1 To whom correspondence should be addressed. Fax: (614) 424-5263. E-mail: hejtman{at}battelle.org. 
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