CYTOTOXICITY OF COLCHICINE DERIVATIVES IN PRIMARY CULTURES OF HUMAN HEPATOCYTES

BACKGROUND
Colchicine has been used to treat gout for centuries. However, owing to its toxicity it displays a variety of side effects. The replacement of colchicine by less toxic but still active derivatives would solve this drawback.


AIM
The aim of this study was to examine the cytotoxicity of 17 colchicine derivatives.


METHODS
Primary cultures of human hepatocytes were the model of choice. Prior to testing, we measured the biochemical parameters of liver donors and the toxicological response of the hepatocytes cultures. For toxicity testing, cells were treated for 24 h with tested compounds in concentrations 1-100 microM. We monitored lactate dehydrogenase (LDH) leakage into the medium, mitochondrial activity (MTT test) and secretion of albumin.


RESULTS
Our data show that LDH and MTT were less sensitive parameters compared to albumin secretion for monitoring the toxicity of colchicine derivatives. Compounds with lower antimitotic activity displayed lowered toxicity.


CONCLUSION
Since human hepatocytes in culture are quiescent cells, they are not as susceptible to tropolone alkaloids as proliferating cells. Screening for colchicine derivatives with lowered cytotoxicity revealed that 10-O-demethylated compounds might be the substances of choice.


INTRODUCTION
Colchicine has been used to treat gout for centuries.It has also medicinal applications in the treatment of some auto-immune and dermatologic disorders 1,2 .However, owing to its toxicity it displays a variety of side eff ects 1 .Replacement of colchicine by less toxic but still active derivatives would solve this drawback 3 .In addition, structural modifi cation to colchicine could produce compounds with novel pharmacological activity.
At a molecular level, colchicine has various eff ects on tubulin, the major one being a change in the secondary structure of the protein caused by binding to a high affi nity site on the tubulin heterodimer.This binding induces partial unfolding of the secondary structure of βtubulin at the carboxy terminus 4 .This unfolding disrupts the protein regions necessary for microtubule formation 5 .Microtubule disarray leads to a variety of consequences at the molecular level [6][7][8] .
The aim of this study was to examine the cytotoxicity of 17 colchicine derivatives.These were newly synthesized and are not commercially available.The cytotoxicity assessment was performed on primary cultures of human hepatocytes, as the most suitable model for biochemical and toxicological studies on hepatic metabolism in man 9,10 .

Chemicals
Cell culture media, supplements and foetal calf serum were purchased from Sigma (St. Louis, MO, USA).All other chemicals and reagents were of the highest quality commercially available.

Primary cultures of human hepatocytes
Liver samples were obtained from multi-organ donors.The tissue acquisition protocol was in accordance with the requirements issued by the Ethics Commission of the Faculty Hospital Olomouc.The characterization of liver donors is shown in Table 2. Hepatocytes were isolated according to a published protocol 12 .The cells were plated on collagen coated culture dishes using cell density 14 x 10 4 cells/cm 2 .The culture medium used was as described 13 enriched for plating with 5% foetal calf serum (v/v).The medium was exchanged for a serum-free medium four to six hours later and the culture was allowed to stabilize for an additional 24 h.Cultures were maintained at 37°C in 5 % CO 2 humidifi ed incubator.

Cytotoxicity assessment
Hepatocytes were incubated 24 h with the tested compounds.In parallel, cultures were treated with dimethylsulfoxide (DMSO) and 1% v/v Triton X-100 to assess the minimal and maximal cell damage, respectively.Lactate dehydrogenase (LDH) leakage into the medium and MTT assay were measured as the indicators of membrane damage and viability, respectively 14,15 .Albumin secretion in the medium was monitored by radio-immune assay (ALBUMIN RIA kit; IMMUNOTECH a.s., Prague; cat 3251).Cells treated with Triton X-100 represented 100 % LDH activity in the medium; cells treated with DMSO represented 100 % metabolic activity in the MTT assay and 100 % of functionality in albumin secretion assay.The response of primary cultures to Triton X-100 (LDH) is shown in Table 3.

Characterization of liver donors
Liver donors were subjected to virological and biochemical testing prior to the surgery.All donors involved in this report were negative for HIV (human immune-deficiency virus), HCV (hepatitis C virus), EBV (Epstein-Barr virus) and CMV (cytomegalovirus).We also monitored biochemical parameters; i.e.ALT (alanine aminotransferase), AST (aspartate aminotransferase), ALP (serum alkaline phophatase) and Bi (serum bilirubin).Information on the sex, age and biochemical parameters of liver donors are shown in Table 2.We observed no clear relation between biochemical parameters, age and sex of the donors and the quality of liver specimens or freshly isolated hepatocytes.

Characterization of primary human hepatocytes
Following the isolation of human hepatocytes we determined their viability using the Trypan Blue exclusion test (Table 3).Cultures were allowed to stabilize for 24 h.Thereafter, we assessed the "toxicological range -TR" of the cultures, i.e. the responsiveness of the cultures membrane damage by Triton-X.TR was calculated as the ratio of LDH activity in the medium released by cells   challenged with Triton-X over the LDH activity in the medium of control cells (TR = LDH Triton /LDH Control ).The toxicological range spanned between cca 2.5 -11; the differences were more due to the variability of LDH activity released by control cells than to the eff ects of Triton-X (Table 3).

Cytotoxicity of tested compounds
To examine the cytotoxicity of tested compounds in primary cultures of human hepatocytes, we measured the following parameters: (i) lactate dehydrogenase activity in culture medium as an indicator of cell membrane damage; (ii) MTT test as the marker of mitochondrial activity; (iii) secretion of albumin into the medium to monitor hepatospecifi c functions.
Increased activity of LDH in the medium was observed in cultures incubated with compounds A0, A2, A13 and A15 in concentrations as high as 100 μM (Table 4).Similar results were obtained when monitoring the mitochondrial activity of hepatocytes -MTT (Table 5).Tropolone alkaloids are highly toxic compounds.Hence, it seems that LDH and MTT are not optimal and sensitive parameters for monitoring the cytotoxicity of these compounds.This may be due to: (i) Cell membrane and mitochondria are not primary targets for tropolone alkaloids within the cell; (ii) Tropolone alkaloids inhibit Table 3. Characterization of primary human hepatocytes.Viability of freshly isolated hepatocytes was assayed by Trypan blue staining.Cells treated with Triton X-100 represented 100 % LDH activity in the medium (100 % damage); cells treated with DMSO represented basal activity (0 % damage).Toxicological range was calculated as LDH triton /LDH control .In contrast, secretion of albumin was a reliable parameter for monitoring the cytotoxicity of the studied compounds.Intact cytoskeleton is essential for albumin secretion even in non-proliferating cells, i.e in human hepatocytes.Colchicine and its derivatives inhibited the secretion of albumin in a dose-dependent manner.These eff ects were already marked in 1 μM concentrations and the inhibition potency correlated with the ability of compounds to inhibit tubulin polymerization (Table 6).For instance 10 μM colchicine (A0) decreased albumine secretion down-to 28 % of control value, whereas 10-O-demethylcolchicine (colchiceine; A11) at the same concentration had no eff ect on albumin secretion.We have previously reported this phenomenon elsewhere 3 .
In conclusion, the biological activity of colchicine and its derivatives is tightly bound to their inhibitory eff ects on tubulin polymerization.The majority of side eff ects of colchicine is demonstrated in proliferating cells, e.g.intestinal mucosa (nausea, vomiting) or hairs (alopecia) 1 .This implies that replacement of colchicine by derivative with lower anti-tubulin activity will result in lower side effects but also in lower therapeutic eff ects.However, this is only a hypothesis.Our data show that 10-O-demethylated derivatives of colchicine (A11, A12) display signifi cantly lower cytotoxicity in human hepatocytes compared to colchicine.In addition, the tested derivatives may have distinct biological activity which is primarily due to interference with microtubules.

Table 1 .
The list of colchicine derivatives.

Table 2 .
Characterization of liver donors.

Table 4 . Eff ects of tested compounds on LDH activity in culture medium.
Cells were treated 24 h with tested compounds, DMSO (vehicle) and/or Triton X-100.The data are expressed as mean ± SD for fi ve diff erent hepatocyte cultures.In each culture, LDH activity was measured in triplicates.* = value signifi cantly diff erent from DMSO-treated cells (p < 0.05).% of cellular damage was calculated: % damage = 100*(LDH compound -LDH DMSO )/(LDH Triton -LDH DMSO ) Cytotoxicity of colchicine derivatives in primary cultures of human hepatocytes

Table 5 .
Eff ects of tested compounds on MTT.Cells were treated 24 h with tested compounds, DMSO (vehicle) and/or Triton X-100.The data are expressed as mean ± SD for fi ve diff erent hepatocyte cultures.In each culture, MTT was measured in six parallel samples.* = value signifi cantly diff erent from DMSO-treated cells (p < 0.05).The results are presented as % of control (DMSO-treated) value.% of control was calculated: % of control = MTT compound /MTT DMSO

Table 6 . Eff ects of tested compounds on albumin secretion
. Cells were treated 24 h with tested compounds, DMSO (vehicle) and/or Triton X-100.The data are expressed as mean ± SD for two diff erent hepatocyte cultures.In each culture, albumin secretion was measured in triplicates.* = value signifi cantly diff erent from DMSO-treated cells (p<0.05).The results are presented as % of control (DMSO-treated) value.% of control was calculated: % of control = ALBUMIN