Effects of benzo[a]pyrene and lead either in separation or in combination on neuronal survival rate and nuclear DNA damage in vitro

Baijie TU; Feng WANG; Tangchun WU

ABSTRACT

BACKGROUND:

Benzo[a]pyrene injures central and peripheral nerves at a certain degree. The study is at an initial phase concerning to the neurotoxicity of benzo[a]pyrene allied with other toxic objects.

OBJECTIVE:

Molecular biological technique integrated with neuron culture were applied in the study of benzo[a]pyrene and lead applied separately or in combination on neurotoxicity and nuclear DNA damage in vitro.

DESIGN:

Repeated measure.

SETTING:

Teaching-Research Room of Labor and Hygiene of Chongqing Medical University and Laboratory of Thermobiology and Molecular Toxicology of Occupation Medical Institute of Tongji Medical College of Huazhong University of Science and Technology.MATERIALS The experiment was performed in Laboratory of Thermobiology and Molecular Toxicology of Occupation Medical Institute of Tongji Medical College ofHuazhong University of Science and Technology from June to September 2003. Ten SD rats of 8-day old were employed and their brain tissues were prepared as primary cell culture object, which were divided into 10 culture groups, 5 culture dishes in each group. The managements were as follows in each group [1] blank control; [2] solvent control[parallel management with equivalent dimethyl sulfoxide (DMSO) +liver microsome enzyme]; [3] lead group of low concentration (acetic lead 5 μmol/L) (No. 1 group); [4] lead group of high concentration (acetic lead 50 μmol/L) (No. 2 group); [5] benzo[a]pyrene group of low concentration (benzo[a]pyrene 5 μmol/L + liver microsome enzym) (No. 3 group); [6]benzo[a]pyrene group of high concentration (benzo[a]pyrene 50 μmol/L +liver microsome enzym) (No. 4 group); [7] lead of low concentration +benzo[a]pyrene of low concentration group (No. 5 group); [8] lead of low concentration + benzo[a]pyrene of high concentration group (No. 6 group);[9] lead of high concentration + benzo[a]pyrene of low concentration group (No.7 group); [10] lead of high concentration + benzo[a]pyrene of high concentration group (No. 8 group).

METHODS:

After stained poisoning for 90 minutes, trypsin digestion method was used for sample collection and trypan blue staining method was applied to assay cell survival rate in each group. Single-cell gel electrophoresis (SCGE) was used to determine the damage of nuclear DNA in each group.MAIN OUTCOME

MEASURES:

Neuronal survival rate and damage rate of nuclear DNA in poisoning with benzo[a]pyrene and lead either in separation or in combination.

RESULTS:

[1] Cell survival rates in various groups poisoned with benzo [a]pyrene and lead of two concentrations either in separation or combination were lower than the controls [poisoning group (44.14±4.80)% to(82.40±2.70)%, the controls (88.44±2.53)% to ( 90.12±2.23)%, P ＜ 0.05to 0.01]. [2] Degrees of nuclear DNA damage in single poisoning group with benzo[a]pyrene of high concentration, lead of low concentration + benzo[a]pyrene of high concentration, lead of high concentration + benzo[a]-pyrene of low concentration and lead of high concentration + benzo[a]pyrene of high concentration were higher than the controls [63% (19/30), 87%(26/30), 80% (24/30), 97% (29/30), 13% (4/30), 20% (6/30), P ＜ 0.01].

CONCLUSION:

Both benzo[a]pyrene and lead present neurotoxicity in vitro and coordinate with each other. The damage of benzo[a]pyrene is worse than lead in neuronal nuclear DNA cultured in vitro.