doi:10.1016/j.taap.2005.07.003 
Copyright © 2005 Elsevier Inc. All rights reserved.
In vivo assessment of toxicity and pharmacokinetics of methylglyoxal Augmentation of the curative effect of methylglyoxal on cancer-bearing mice by ascorbic acid and creatine
Manju Ghosha, Dipa Talukdara, Swapna Ghosha, Nivedita Bhattacharyyaa, Manju Raya, 
, 
and Subhankar Rayb
aDepartment of Biological Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700 032, India
bDepartment of Biochemistry, University College of Science, University of Calcutta, Kolkata-700 019, India
Received 7 April 2005;
revised 24 June 2005;
accepted 2 July 2005.
Available online 19 August 2005.
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Abstract
Previous in vivo studies from several laboratories had shown remarkable curative effect of methylglyoxal on cancer-bearing animals. In contrast, most of the recent in vitro studies have assigned a toxic role for methylglyoxal. The present study was initiated with the objective to resolve whether methylglyoxal is truly toxic in vivo and to reassess its therapeutic potential. Four species of animals, both rodent and non-rodent, were treated with different doses of methylglyoxal through oral, subcutaneous and intravenous routes. Acute (treatment for only 1 day) toxicity tests had been done with mouse and rat. These animals received 2, 1 and 0.3 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Chronic (treatment for around a month) toxicity test had been done with mouse, rat, rabbit and dog. Mouse, rat and dog received 1, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. Rabbit received 0.55, 0.3 and 0.1 g of methylglyoxal/kg of body weight in a day through oral, subcutaneous and intravenous routes respectively. It had been observed that methylglyoxal had no deleterious effect on the physical and behavioral pattern of the treated animals. Fertility and teratogenecity studies were done with rats that were subjected to chronic toxicity tests. It had been observed that these animals produced healthy litters indicating no damage of the reproductive systems as well as no deleterious effect on the offspring. Studies on several biochemical and hematological parameters of methylglyoxal-treated rats and dogs and histological studies of several organs of methylglyoxal-treated mouse were performed. These studies indicated that methylglyoxal had no apparent deleterious effect on some vital organs of these animals. A detailed pharmacokinetic study was done with mouse after oral administration of methylglyoxal. The effect of methylglyoxal alone and in combination with creatine and ascorbic acid on cancer-bearing animals had been investigated by measuring the increase in life span and tumor cell growth inhibition. The results indicated that anticancer effect of methylglyoxal was significantly augmented by ascorbic acid and further augmented by ascorbic acid and creatine. Nearly 80% of the animals treated with methylglyoxal plus ascorbic acid plus creatine were completely cured and devoid of any malignant cells within the peritoneal cavity.
Keywords: Methylglyoxal; Ascorbic acid; Creatine; Toxicity; Pharmacokinetics; Cancer treatment
Fig. 1. Histological examination of different organs of mouse, both untreated (control) and treated orally by methylglyoxal. (A and B) Liver, control and treated respectively; magnification 40×. (C and D) Kidney, control and treated respectively; magnification 10×. (E and F) Spleen, control and treated respectively; magnification 40×. (G and H) Duodenum, control and treated respectively; magnification 10×. (I and J) Bone marrow, control and treated respectively; magnification 100×. The stain used for bone marrow was Leishmann, for other organs, hematoxylin and eosin.
Fig. 2. .Blood methylglyoxal concentrations in mouse after single oral dose of methylglyoxal. ○, ●, Δ and □ represent 0, 50, 100 and 200 mg of methylglyoxal respectively.
Fig. 3. Blood methylglyoxal concentrations in mouse in repeat oral dose study.
Fig. 4. Effect of methylglyoxal, methylglyoxal plus ascorbic acid and methylglyoxal plus ascorbic acid plus creatine on EAC cell-inoculated mice. Photographs were taken of animals one from each group, EAC cell counts of which are presented in Table 9. Details of inoculation with EAC cells and treatment schedule are described in the legend of Table 9. The photographs were taken on day 18. (A) Normal mouse. (B) Control animal (EAC cell-inoculated, without any treatment). (C) Treated with MG 30 mg. (D) Treated with MG 30 mg + AA 50 mg. (E) Treated with MG 30 mg + AA 50 mg + 150 mg. The drugs applied were /kg body weight/day.
Table 1.
A summary protocol of mode and dose of treatment of methylglyoxal for different studies
Duration of the treatment is described in the text.
a n = number of animals in each group × number of groups.
Table 2.
Long-term (chronic) toxicity in animals: measurement of body weight
Amount of methylglyoxal received by each animal: mouse and rat- 1gm (oral), 0.3 gm (subcutaneous) and 0.1 gm (intravenous); rabbit –0.55 gm (oral), 0.3 gm (subcutaneous) and 0.1 gm (intravenous). Total number of animals in each group including control and different modes of treatment: 6 (mouse), 5 (rat) and 4 (rabbit). Each set of experiment was repeated 4 times for mouse and rat and for rabbit 2 times respectively. For each mode of treatment a similar study with lesser amount of methylglyoxal was done for mouse and rat and similar results were obtained.
a Weight of animals for mouse and rat in gm and for rabbit in Kg.
Table 3.
Biochemical tests of blood/sera of rats
Table 4.
Effect of methylglyoxal treatment on the level of several marker enzymes and metabolites of sera and on cell population of blood of dog and bitch
(A – dog, B – bitch, C – dog).
n.d. : not determined.
Table 5.
Pharmacokinetic parameters of methylglyoxal in mice after a single oral dose of methylglyoxala,b
a Values are means.
b Two mice were used in each dose level.
c Abbreviations: C
max – maximum blood concentration, t
max – time to C
max, k
a– apparent absorption rate constant, V—apparent volume of distribution, CL—apparent total body clearance, k – elimination rate constant, t
1/2 – elimination half-life.
Table 6.
Pharmacokinetic parameters of methylglyoxal in blood of mice dosed orally by 100 mg/kg body wt./ day of methylglyoxal
Abbreviations and symbols are similar to Table 5.
Table 7.
Increase in life span of EAC cell-inoculated mice treated with methylglyoxal ascorbic acid and creatine
For this experiment, from a total number of 105 mice, 15 animals received a particular mode of treatment. Control group received only normal saline; the other five groups received methylglyoxal alone or methylglyoxal plus ascorbic acid or methylglyoxal plus ascorbic acid plus creatine. All the test substance was dissolved in 0.9% NaCl and 0.5 ml was separately injected intraperitoneally once day for consecutive 14 days. The day on which 105 EAC cells were inoculated into each mouse was considered as day 0. The treatment started from day 3. The amount of each compound indicated in the parentheses is the amount administered per kg body weight per day.
MG – Methylglyoxal; AA – Ascorbic acid, Cr- Creatinine.
a Creatine was dissolved in water and the mice were fed instead of injected.
Table 8.
Increase in the percentage of body weight of the EAC cell inoculated mice receiving different treatments
For this experiment, from a total number of 36 animals, 6 animals received a particular mode of treatment. The details of the inoculation and treatment schedule were identical to Table 7. The body weight was measured from day 15. The amount of each compound indicated in the table is mg per kg body weight per day.
MG – Methylglyoxal, AA – Ascorbic acid, Cr – Creatinine. a – 2 animals survived out of 6, b – 2 animals survived out of 6, c – 3 animals survived out of 6.
Table 9.
Tumor growth inhibition study
Each group consists 10 mice. Each mouse was inoculated with 105 (0.1 million) EAC cells. Treatment with three different drug combinations were started from day 3 and continued up to day 16. The day of inoculation was considered as day 0. The mode of treatment of the drugs was similar to that of Table 7. The ascites fluid containing cells were quantitatively removed from the peritoneal cavity on the indicated day and cell volume and number were counted. The details of collection and counting are described in Materials and Methods. The amount of each drug as indicated in the table is mg per kg body weight per day.
d – Dry; n.d. – Not detectable; MG – Methylglyoxal; AA – Ascorbic acid, Cr – Creatinine.
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