Artículo 1 Toxicidad in vitro

Introduction

In early stages of drug discovery, in vitro toxicity evaluation is very important to obtain
quick evaluation with small amount of test compounds and reduce animal use. Primary
rat hepatocytes are widely used for in vitro hepatotoxicity evaluation. The cytotoxic
concentrations in rat hepatocytes are different from that in cell lines such as HepG2,
HeLa, and BALB/c 3T3 especially in compounds in which metabolites have
hepatotoxicity. Human hepatocytes are also used for in vitro hepatotoxicity evaluation,
but maintaining viability and liver-specific functions is difficult because of
cryopreservation and shipping. Primary culture of rat cardiomyocytes has been used for
in vitro caridiotoxicity evaluation. Neonatal, rather than adult, rat cardiomyocytes have
been used because they are easy to disperse and have higher viability after isolation and
culturing. These in vitro evaluations are usually performed separately for each organ;
parallel assay in different organs is not widely carried out. In this investigation, to
establish a predictive in vitro cardiovascular toxicity and hepatotoxicity screening
system, whole cells from the neonatal rat heart and rat hepatocytes were cultured with
known cardiotoxic and hepatotoxic compounds and cytotoxicities (LDH contents) were
compared.

Materials and methods

Chemicals: Epirubicin (EPI), daunorubicin (DNR), idarubicin (IDA), doxorubicin
(DOX), acetaminophen (APAP), cyclophosphamide (CPA), diclofenac (DCF),
disulfiram (DSF), lithocholic acid (LCA) were purchased from Sigma (Tokyo, Japan).
Minimal essential medium (MEM), William's E medium, penicillin (10000 U/mL)-
streptomycin (10000 μg/mL) solution, L-glutamine solution (200 mM), HEPES buffer
(1 M, pH 7.3), heat-inactivated fetal bovine serum (FBS), Hank's balanced-salt solution
(HBSS), Ca, Mg-free Hank's balanced-salt solution (HBSS(-)) were purchased from
Invitrogen (Tokyo, Japan).
Neonatal rat heart cell culture: The heart was isolated from neonatal (1 — 5 days old)
Crl: CD (SD) rats (Charles Liver Japan, Tokyo, Japan) and dispersed on a rotation
culture with an enzyme mixture of 2.5 mg/mL trypsin (1:250; Invitrogen), 1.5 mg/mL
collagenase (Wako, Osaka, Japan), and 1.5 mg/mL crystallized bovine albumin
(Serologicals Proteins Inc., IL, USA) in MEM at 37ºC for approximately 60 min. The
dispersed cells were collected every 15 — 20 min and the remaining undispersed tissues
were re-suspended in the enzyme mixture solution and the rotation culture was
continued. The dispersed cells were washed with HBSS(-) and suspended in MEM
supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-
glutamine, and 10 mM HEPES (pH 7.3). The cell number was adjusted to 3×105/mL
and cultured in 96-well plates on pre-formed type I collagen gel (BD, Tokyo, Japan)
with 10% Matrigel (BD). After several days of preculture, the supernatant was removed
and the culture medium with a test compound was added to a well and incubated at
37ºC for 24 or 48 hr. After incubation, lactate dehydrogenase (LDH) activity in the cells
(after lysing with 0.05% Triton-X) was measured using a LDH kit (Wako). IC50 values
were calculated from the concentration-response curve as the concentration of a test
compound with a decrease in absorbance equivalent to 50% of the control value.
Rat hepatocyte culture: The hepatocytes were isolated by two-step in situ perfusion
with collagenase from 6 — 10-week-old SD rats as previously described (Seglen, 1976,
Wang, 2002). Briefly, the liver was perfused with 0.5% EGTA (Dojindo, Kumamoto,
Japan) in HSBB(-) from the portal vein for approximately 5 min and then perfused with
0.05% collagenase + trypsin inhibitor (Sigma) in HBSS for 8 — 10 min. The dispersed
cells were washed with ice-cold HBSS(-) and suspended in William's E medium
supplemented with 10% FBS, 0.1 μM insulin (Sigma), 1 μM dexamethasone (Sigma),
100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM L-glutamine, and 10 mM HEPES
(pH 7.3). The cell number was adjusted to 3× 104 cells/mL and cultured in 96-well
plates coated with type I collagen (IWAKI, Tokyo, Japan). After preculture for several
hours, the supernatant was removed and the culture medium with a test compound was
added and incubated at 37ºC for 24 hr. After incubation, LDH activities in the cells
were measured. IC50 values were calculated from the concentration-response curve as
the concentration of a test compound with a decrease in absorbance equivalent to 50%
of the control value.

Results
Cuestionario Artículo 1

1. Defina el (los) objetivo (s) de la investigación presente. ¿Qué experimentos se

llevan a cabo para darle cumplimiento?

2. ¿A qué rama de la Toxicología pertenece el método utilizado en el artículo?

¿Qué otros ejemplos usted conoce?

3. ¿Por qué cree usted que se utiliza la LDH como marcador de citotoxicidad?
¿Qué otros marcadores pudieran utilizarse?

4. Defina en valor de Concentración Inhibitoria Media (CI50). ¿Por qué cree usted
que se comparan los resultados con respecto a este valor?

5. Describa y explique los resultados mostrados en los gráficos 1 y 2. ¿Cuál

compuesto resulta más toxico y cuál menos tóxico?

6. Analice los resultados mostrados en la Tabla 1.

a) ¿Qué importancia tiene que se comparen los resultados obtenidos en el

artículo con los que se han obtenido en modelos in vivo?
b) ¿Qué desventajas cree usted que tiene comparar resultados obtenidos en
modelos in vitro con los obtenidos en modelos in vivo?
c) Según los resultados mostrados en la columna 1 de esta tabla: ¿Existe una
buena correlación entre los resultados in vitro y los obtenidos in vivo?
Formule una conclusión parcial.

7. Analice los resultados mostrados en la Tabla 2. ¿Qué significa la relación IC 50

heart/ IC50 hepatocyte sea menor que 1 en todos los casos? Formule una
conclusión parcial.

8. Analice los resultados mostrados en la Tabla 3. ¿Por qué es necesario realizar

experimentos donde se evalúen compuestos hepatotóxicos? Analice la relación
IC50 heart/ IC50 hepatocyte. Formule una conclusión parcial.

9. ¿Cuáles serían para usted las consideraciones finales de la investigación? ¿Cuál

es la conclusión más importante?

10. Si usted fuera a estudiar por vez primera la toxicidad en cierta droga:

a) ¿De qué órganos haría cultivos celulares para evaluar la toxicidad? ¿Por
qué?
b) ¿Qué ventajas presentan los modelos in vitro en las evaluaciones
toxicológicas?