Microsoft word - sochacka
THE EFFECT OF CULTURE CONDITIONS ON TOXICITY OF
6-MERCAPTOPURINE TO CHLORELLA VULGARIS
Department of General and Analytical Chemistry, Medical University of Silesia,
Faculty of Pharmacy, Jagiellonska 4, 41-200 , Sosnowiec, Poland
The thiopurine antimetabolite 6-Mercaptopurine (6-MP) is an analogue of the
purine base hypoxanthine and is indicated for remission induction and maintenance
therapy of acute lymphatic leukemia. The active metabolites of 6-MP alter cellular metabolism in a number of ways, including inhibition of purine biosynthesis de novo
and incorporation into cellular RNA and DNA .
6-MP is practically insoluble in water, it dissolves in dilute solutions of alkali
hydroxides and hydrochloric acid. The ionization constants, pK,
for anion formation is 7.7 (in pyrimidine ring) and 11.8 (in imidazole ring) and for cation formation < 2.5 (in
imidazole ring) . The partition coefficient octanol-water, log P
ow, is -0.19 . 6-MP
can exist in six tautomeric forms because of the protropic tautomerism of the imidazole
ring and thione-thiol tautomerism of the pyrimidine ring. 6-MP in aqueous solution may exist in the equilibrium of tautomeric forms, but in alkaline medium is practically
Figure 1. Structures of the neutral and anionic forms of 6-Mercaptopurine.
An adsorption and distribution of the drug are determined by its
physicochemical properties e.g. its ionization and a partition coefficient between lipid
and aqueous phases corresponding to drug lipophilicity. Non-ionized form of the drug is most often preferably dissolved in lipids and thus is easily transported through lipid
In recent studies about toxicity of new synthesized purine thioderivatives
relative to Chlorella vulgaris
, in which 6-MP was treated as reference compound, cultures were processed in culture media with pH of 6.7 – 7.0 [4, 5]. However C. vulgaris
exhibit high resistance to pH changes of culture medium (even to 11.0). In this work the effect of increased pH on 6-MP toxicity was examined.
Experiments were carried out with unicellular green alga Chlorella vulgaris
Beij., strain 264, Boehm and Borns 1972/1 CCALA (Czechoslovak Academy of Science). The cultures were conducted in 250 ml Erlenmeyer flasks with 50 ml of
modified Kuehl-Lorenzen  liquid medium (enriched with 4% CO2 and 3.85 mmol/l HCO -
3 ) at 24 ± 2oC and with continuous mixing using a magnetic stirrer. The cultures
were synchronized by light-dark cycles (12-h light and 12-h dark period). The 6-
Mercaptopurine (Sigma Aldrich) was added to algal medium as a solution in DMSO or 0.1 mol/l NaOH to obtain the required concentration (48.0, 96.0 and 144.0 mg/l). The
final concentration of DMSO and NaOH did not inhibit nor stimulated the growth of C. vulgaris
. The initial pH of the liquid medium was 6.7, 7.7 or 9.0. The tested cultures
and control cultures (without the 6-MP) were conducted in parallel. Three replicates of each examined concentration and six replicates of the controls were examined. A
growth of the cultures was monitored after 24 and 48 hs by measuring spectrophotometrically the optical density of the cell suspension at 680 nm. Growth
inhibition and EC50 were calculated according to ISO 8692 .
The distribution coefficient, log D
, which is a function of both its lipophilicity
when non-ionized and the degree of ionization and is defined as the effective lipophilicity of a compound at a given pH, was calculated using eq. (1) :
) – 1 = pK
– pH (1) where: pK
= 7.72 , log P
= 0.62 is the value calculated for neutral form of 6-MP from
experimental values obtained with RPTLC method .
The amount (%), of 6-MP ionized form were calculated using the eq. (2) :
% ionized = 100/[1 + 10(pK
– pH)] (2)
Absorption spectra of the 6-MP in standard solutions of pH 5.0 – 13.0 and in
liquid media (after the end of the cycle) were recorded between 220 and 380 nm using Jasco (V-530) UV VIS Spectrophotometer.
The calculated distribution coefficient (log D
) and the percentage of 6-MP
ionized form in solutions of pH range 5.0 – 9.0 are presented in Table 1. The presented
results show that effective lipophilicity of 6-MP is depended to solution pH and will decrease if pH and amount of ionized form increases.
Table 1. The effect of pH on the physicochemical properties of 6-MP
The λmax values of 6-MP standard solutions in a pH range of 5.0 – 13.0
determined according to UV spectra calibration curves (ultraviolet spectra) are also presented in Table 1. The changes of UV maximum absorption and hypsochromic shift of λmax of about 13 nm for 6-MP solutions are related to formation of monoanionic form
from neutral one and dianionic form from monoanionic one.
The characteristic of C. vulgaris
culture growth is presented in Table 2 as
percentage of growth inhibition related to control culture. In the same table the λmax
values determined for 6-MP in a culture media are presented that confirms various ionization state of this compound. The determined values of 6-MP toxic concentration
related to C. vulgaris
are expressed as EC50 (mg/l). These results indicate that toxicity of 6-MP is depended to pH of culture media. The EC50 value for culture media of pH
9.0 was twice higher than one determined for culture media of pH 6.7.
Table 2. EC50 values characterizing toxicity of 6-MP to C. vulgaris
% inhibitiona λmaks [nm] of 6-MP in the liquid medium
a inhibition percentage related to the control culture
Obtained results indicate that pH of culture media influences the toxicity of 6-
MP and the ionized forms of 6-MP were significantly less toxic for C. vulgaris
phenomen may be caused by decreased lipophilicity of anionic form relative to neutral one. Lipophilicity is an important property affecting the bioactivity of drugs and
increasing lipophilicity usually correlates with increasing biological activity. The transport rate and the amount of transported substance into the cell is positively
correlated with a partition coefficient log P
. The log P
depends on pH and degree of dissociation, which directly corresponds to the amount of dissociated form. Non-ionized
and lipophilic substance is easily transported into the cell because the cell membrane is a selectively permeable lipid bilayer. However, it has only a very low permeability to
ionic molecules and thus their concentration inside the cell will be lower.
The other cause of decreased toxicity of dissociated 6-MP might be its impaired
interaction with DNA. 6-MP is an antimetabolite of natural purine base and may be
incorporated into DNA as deoxy-6-thioguanosine (S6G) instead of guanine and thus modify structural properties of DNA duplex.
Bohon J.  and Somerville L.  have shown, that active metabolite S6G (in
keto form) was incorporated into DNA. This required formation of weakened Watson-
Crick hydrogen bonds which led to S6G-C and S6G-T pairs (Fig. 2). However, S6G-C is highly favoured because of triple hydrogen-bond.
It can be concluded, that 6-MP monoanionic form generated during N(1)-H
dissociation at pH 7.7 and 9.0 may not combine with natural base by forming Watson-
Crick hydrogen bonds. This is a reason why this form S6G does not incorporate into DNA duplex and, in consequence, does not show cytotoxic activity to C. vulgaris
Figure 2. Base pair configuration for S6G-C, S6G-T and G-T and canonical G-C . REFERENCES
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Environmental Toxicology and Chemistry, Vol. 32, No. 12, pp. xx–xx, 2013ACUTE AND CHRONIC EFFECTS OF DIPHENHYDRAMINE ANDSERTRALINE MIXTURES IN CERIODAPHNIA DUBIAERIC W. GOOLSBY,y CHASE M. MASON,z JAMES T. WOJCIK,y ALEX M. JORDAN,y and MARSHA C. BLACK*yyDepartment of Environmental Health Science, University of Georgia, Athens, Georgia, USAzDepartment of Plant Biology, University of Georgia
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