Abstract 4933

Background:

Curcumin (diferuloylmethane) is an active ingredient of turmeric and has been suggested to have antiproliferative, pro-oxidant and cytotoxic effects (Das R, Apoptosis 2008). Curcumin has also been shown to have antioxidant effects (Rastogi M, Free Radic Res 2008). Our aim was to assess the cytotoxic, pro-oxidant and antioxidant effects of curcumin on rat peripheral blood lymphocytes (RPL) in culture.

Methods:

Different concentrations of curcumin (0.325 μM, 0.65 μM, 1.3 μM) were analyzed for cytotoxic effects on rat peripheral lymphocytes. The cytotoxic effect of curcumin was assessed by measuring cell viability using Trypan blue exclusion assay. DNA damage by curcumin (at 0.65 μM) was confirmed by the COMET assay. The effect of curcumin (at 0.65 μM) on oxidation was assessed by measuring the activity of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and lipid peroxidation (LP) by spectrophotometry. RPLs were cultured for 72 hr in vitro. The pro-oxidant effect of curcumin was determined by comparing LP induction in control (PBS treated) vs curcumin treated RPLs. Parameters were measured at 0, 24 and 48 hrs of curcumin exposure. Reactive oxygen species (ROS) generated by treatment with FeCl3 (10 mM) and H2O2 (5 mM) (Fe/H) were used to demonstrate the ROS scavenging effect of curcumin. Lymphocyte cultures were treated with either Fe/H alone or in combination with Curcumin. Oxidation levels were assessed as described above at 0, 24, 48 hrs following exposure.

Results:

Cytotoxic effects were seen at different curcumin concentrations (Table 1). Viability of RPLs were 42% at 0.64 μM concentration vs 72% at 0.325 μM. 30% decrease in cell viability of RPLs were seen at 0.65 μM concentration compared to 0.325 μM. Hence, 0.65 μM concentration was used for further experiments. There was a significant increase in DNA damage in curcumin exposed lymphocytes at 48hr (table 2). There was a significant increase in LP in curcumin exposed RPLs compared to control lymphocytes. By contrast, there was no significant change in LP values in presence of Fe/H at all time points compared to control. There was a significant decrease in LP when RPL cultures were incubated with curcumin for 48 hr compared to 0 hr of incubation. There was significant increase in activities of SOD, catalase and GPx in presence of curcumin and Fe/H (table 3 and 4).

Table 1:

Cytotoxic effect of curcumin as demonstrated Trypan blue viability assay Cnt=control

Lymphocyte cultureLymphocyte cell viability at 48 hr (%)
Cnt 84 
Curcumin 0.325 μM 72 
Curcumin 0.65 μM 42 
Curcumin 1.3 μM 35 
Lymphocyte cultureLymphocyte cell viability at 48 hr (%)
Cnt 84 
Curcumin 0.325 μM 72 
Curcumin 0.65 μM 42 
Curcumin 1.3 μM 35 
Table 2:

Cytotoxic effect of curcumin (at 0.65 μM) as demonstrated by % Tail DNA. Two sample t-test was used to calculate p value

% tail DNA (mean ± SD)
Cnt (at 48 hr) (n= 6) 0.53 ± 0.71 
Curcumin exposed lymphocyte (at 48 hr) (n=4) 32.96 ± 17.97 
Difference in % tail DNA between control and Curcumin at 48 hrs p = 0.0019 
% tail DNA (mean ± SD)
Cnt (at 48 hr) (n= 6) 0.53 ± 0.71 
Curcumin exposed lymphocyte (at 48 hr) (n=4) 32.96 ± 17.97 
Difference in % tail DNA between control and Curcumin at 48 hrs p = 0.0019 
Table 3:

Activities of antioxidant enzymes- SOD, catalase, GPx and LP level in optical density (OD- mean of 3 set of cultures)

Lymphocyte culture (C) ODOD at 0hrOD at 24hrOD at 48hr
Prooxidant Activity (n=3)  Only curcumin 
LP 0.0310 0.1130 0.0850 0.05067 
Antioxidant activity (n=3) PBS+ FeCl3+H2O2 Curcumin. + FeCl3+H2O2 
LP 0.0220 0.0250 0.0160 0.01266 
SOD 0.0560 0.0230 0.0540 0.13333 
Catalase 0.0200 0.0933 0.2230 0.0936 
GPx 0.0120 0.0363 0.1233 0.2170 
Lymphocyte culture (C) ODOD at 0hrOD at 24hrOD at 48hr
Prooxidant Activity (n=3)  Only curcumin 
LP 0.0310 0.1130 0.0850 0.05067 
Antioxidant activity (n=3) PBS+ FeCl3+H2O2 Curcumin. + FeCl3+H2O2 
LP 0.0220 0.0250 0.0160 0.01266 
SOD 0.0560 0.0230 0.0540 0.13333 
Catalase 0.0200 0.0933 0.2230 0.0936 
GPx 0.0120 0.0363 0.1233 0.2170 
Table 4:

Comparisons for enzyme activities between cnt and curcumin. One way ANOVA test was used to derive p values.

NS= no significant

Comparison of enzyme activities between Cnt and Curcumin (at 0, 24, 48 hr)Comparison of enzymes activities (in presence of curcumin and Fe/H) at various time points
 Cnt- 0hr cnt-24hr cnt-48hr 0-24 hr 0-48 hr 
Pro-oxidant effect 
LP <0.0001 <0.0001 <0.0002 NS NS 
Antioxidant effect Culture+ Fe/H (control) Vs Curcumin. + Fe/H Comparison of enzymes activities (in presence of Curcumin and Fe/H) at various time points 
LP NS NS NS NS 0.0392 
SOD NS NS <0.0028 0.0015 <0.0006 
Catalase <0.0053 <0.0001 <0.0051 0.0009 NS 
GPx <0.0249 <0.0001 <0.0001 <0.0001 <0.0001 
Comparison of enzyme activities between Cnt and Curcumin (at 0, 24, 48 hr)Comparison of enzymes activities (in presence of curcumin and Fe/H) at various time points
 Cnt- 0hr cnt-24hr cnt-48hr 0-24 hr 0-48 hr 
Pro-oxidant effect 
LP <0.0001 <0.0001 <0.0002 NS NS 
Antioxidant effect Culture+ Fe/H (control) Vs Curcumin. + Fe/H Comparison of enzymes activities (in presence of Curcumin and Fe/H) at various time points 
LP NS NS NS NS 0.0392 
SOD NS NS <0.0028 0.0015 <0.0006 
Catalase <0.0053 <0.0001 <0.0051 0.0009 NS 
GPx <0.0249 <0.0001 <0.0001 <0.0001 <0.0001 
Conclusions:

Dose dependent cytotoxic and DNA damage effects of curcumin are seen in RPLs. Treatment with curcumin alone increases lipid peroxidation, However in the presence of oxidative stress, curcumin may act as an antioxidant (ROS scavenging effect) by stimulating activities of antioxidant enzyme and preventing lipid peroxidation.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

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Asterisk with author names denotes non-ASH members.