Evaluation of cytotoxicity and mutagenicity of the benzodiazepine flunitrazepam in vitro and in vivo

Flunitrazepam (FNZ) is a sedative benzodiazepine prescribed for the short-term treatment of insomnia. However, there are concerns regarding possible carcinogenic or genotoxic effects of this medicine. Thus, the aim of this study was to evaluate the cytotoxic, clastogenic and aneugenic effects of FNZ in hepatoma cells from Rattus norvegicus (HTC) in vitro and in bone marrow cells of Wistar rats in vivo. These effects were examined in vitro following treatment with 0.2, 1.0, 5.0 or 10 μg/mL FNZ using a micronucleus test with a cytokinesis block or in vivo using a chromosomal aberration test following treatment with 7, 15 or 30 μg/mL/kg body weight. The results showed that the benzodiazepine concentrations tested were not cytotoxic, aneugenic or clastogenic. However, considering the adverse effects of using this benzodiazepine, more studies are required.


INTRODUCTION
Flunitrazepam (FNZ) is a potent benzodiazepine agonist primarily used as a hypnotic and preanesthetic agent across Europe (Woods, Winger, 1997;Druid, Holmgren, Ahlner, 2001).FNZ is a sedative benzodiazepine that is prescribed for the short-term treatment of insomnia at a recommended dose of 0.5-1.0mg, with a maximum dose of 2.0 mg (Ohshima, 2006).FNZ has an appreciable abuse potential (Druid, Holmgren, Ahlner, 2001;Ohshima, 2006) and is among a cluster of "club drugs" misused at raves and in other social settings, including in the United States, where this drug is not legally available (Woods, Winger, 1997;Smith et al., 2002;Britt, Mc-Cange-Katz, 2005).
Similar to other cerebral modulating drugs, FNZ can produce pharmacological effects that include sedation, memory impairment, and behavioral disinhibition, which is described as an increase in the probability of behaviors that typically occur at low frequencies due to social and interpersonal constraints, such as sexual misconduct.These disinhibitory and amnesic effects might result in increased risky behavior and altered decision-making (e.g., violence, other drug use and high-risk sexual activity) (Wu, Schlenger, Galvin, 2006;Lane, Cherek, Nouvion, 2008).
Individuals who abuse FNZ often commit sexual assaults and violent acts (Almeida et al., 2010).The increasing abuse of both FNZ and ketamine hydrochloride has particularly been observed in young people in social settings, such as clubs.Frequently, young people take FNZ in conjunction with alcohol and exhibit euphoria, agitation and amnesia (Druid, Holmgren, Ahlner, 2001;Smith et al., 2002).FNZ is becoming increasingly abused by young people who use this drug in combination with alcohol and then commit violent crimes (Almeida et al., 2010).
In addition to these harmful effects, there are concerns regarding possible genotoxic or carcinogenic effects caused by this medication or its derivatives (Assaf, Abdel-Rahman, 1999;Brambilla, Carrozzino, Martelli, 2007), thereby stressing the importance of cytogenetic testing with FNZ.
Short duration test systems are widely used to detect environmental mutagens, such as ionizing radiation or chemically synthesized substances.These systems include cultures of mammalian cells grown in vitro as well as cultures of human lymphocytes and cell lines from Chinese hamsters and rats (Rattus norvegicus).In turn, in vivo test systems, such as rats, respond in a manner similar to that observed in vitro.Additionally, a close correlation exists between the biological metabolism of rats and that of humans, which justifies the use of this animal in the identification of substances that may possess mutagenic potential in humans (Waters et al., 1996;Natarajan, 2002).
The aim of this study was to evaluate the cytotoxic, aneugenic and clastogenic potential of FNZ through the induction of micronuclei in cultured hepatoma cells in vitro and through the appearance of chromosomal aberrations in R. norvegicus bone marrow cells in vivo.

In vitro
Cell Line -HTC HTC cells that were derived from a Rattus norvegicus hepatoma were provided by Dr. Mário Sérgio Mantovani, State University of Londrina -Paraná -Brazil.Cells (2x10 6 ) were grown in 25 cm 2 culture flasks containing 5 mL DMEM (Gibco), which was supplemented with 10% fetal bovine serum (Gibco), incubated at 37 °C.Accordingly, the lineage cell cycle is approximately 24 hours.

Treatment Solution
For the negative control, 20 µL of phosphate buffered saline (PBS)/mL was added to the culture medium (DMEM).The positive control was doxorubicin (DXR -Ackros) at a final concentration of 0.2 μg/mL DMEM.
All of the experiments were performed in triplicate; 3,000 binucleated cells were analyzed per treatment to assess the frequency of micronuclei (MN) and 1,500 cells were counted to determine the cytokinesis block proliferation index (CBPI).The process for selecting binucleated cells, identifying micronuclei and calculating the CBPI was followed as described by Fenech (2000).The percentage of cytostasis was calculated by the following formula (OECD, 2010): Statistical analysis was performed using an ANOVA and Tukey's test (p < 0.05).

Wistar Rats
Six Wistar rats, three males and three females for each group, were obtained from the Central Vivarium -State University of Maringá (UEM).Experiments were performed on 35 days old rats weighing approximately 100 g body weight (bw).Animals were maintained during the period of experimentation in the Central Vivarium of the Department of Cell Biology and Genetics/UEM under controlled conditions of temperature ± 25 ºC, humidity ± 50% and with a photoperiod of 12 hours light/ dark, according to the standards established by the Ethics Committee on Experimentation with Laboratory Animals/ UEM (process number: PRO 046/2010).

Treatment Solution
For the negative control, rats were treated with 1 mL of water/100 g bw (body weight), via gavage, for 24 hours.The positive control (CO + ) was 1.5 mg of cyclophosphamide clastogenic drug/1 mL water/100 g bw, intraperitoneally administered for 24 hours to assess the responsiveness of the lineage.
Treatments with the benzodiazepine FNZ (Rohypnol®) (Roche) were performed by treating rats with three concentrations of this medicine: 7 [1], 15 [2] or 30 [3] μg/mL of water/kg, via gavage, as calculated by an extrapolation of the concentrations used by humans to the body weight of rats.

Chromosomal Aberration Test
The chromosomal aberration test was performed to obtain bone marrow cells of Wistar rats using the Ford and Hamerton method (1956), with some modifications.Mitotic cells were interrupted in metaphase with the intraperitoneal administration of 0.5 mL/100 g bw of colchicine (0.16%) half an hour before euthanasia.
For animal euthanasia, 0.5 mL/100 g bw of anesthetic (1 g sodium thiopental/25 mL of distilled water) was intraperitoneally administered.
Bone marrow was removed from the femurs with 5 mL of hypotonic solution (0.075 M potassium chloride).The suspension was incubated at 37 °C for 12 minutes, centrifuged for 5 minutes and then the supernatant was discarded.The material was fixed with 5 mL of methanol solution, 3:1 acetic acid and centrifuged for 5 minutes.The supernatant was discarded and the fixative was changed at least twice.The preparation was performed with a drop of suspension on clean slides containing a film of distilled ice water.The coloration of the slides was performed using a drop from a film of Giemsa solution in phosphate buffer (0.12 M Na 2 HPO 4 , x 12 H 2 O and 0.06 M KH 2 PO 4 ) at a ratio of 1:30 at pH 6.8.
The analysis of the slides was performed using a light microscope, analyzing 100 metaphases per animal, which totaled 600 metaphases for the control and treatment groups, and assessing the appearance of alterations, such as gaps, breaks, fragments, etc.The results were expressed as a percentage of total aberrations.
The cytotoxic evaluation of the mitotic index (MI) was calculated from 5,000 cells by sex, totaling 10,000 cells per group.The MI calculation, as a percentage, was performed using the number of dividing cells divided by the total number of cells present in the fields.
Statistical calculation was performed using the chisquare test (a = 0.05).

RESULTS AND DISCUSSION
W h e n e x a m i n i n g t h e c y t o t o x i c i t y o f t h e benzodiazepine FNZ at various concentrations (0.2, 1.0, 5.0 and 10.0 µg/mL) in vitro (Table I), the results showed that this substance did not affect cytokinesis-block proliferation and showed no cytotoxic action in hepatoma cells from R. norvegicus.The addition of FNZ did not cause the inhibition of cell proliferation but stimulated cell proliferation, as shown by the percentage of cytostasis (Table I) at all concentrations evaluated: 11.76% at a dose of 0.2 µg/mL; 10.29% at a dose of 1.0 µg/mL; 11.76% at a dose of 5.0 µg/mL and 14.70% at a dose of 10 µg/mL.
These results differ from those results found by Assaf Abdel-Rahman (1999), who showed that a 0.16 mM solution of FNZ (50 μg/mL) significantly reduced the number of viable rat liver cells after 2 hours of exposure.However, although the base concentration used in this study was extrapolated to the total weight of the culture medium (5 g) based on the highest concentration consumed by humans (2 mg) and although concentrations up to 50 times higher than this concentration were also used, the concentrations used in this study are much smaller than the 50 μg/mL concentration that was used in Assaf Abdel-Rahman's (1999) work, which may explain the lack of cytotoxicity observed in the HTC cells.
Non-cytotoxic results were also observed in vivo in the bone marrow cells of Wistar rats (Figure 2) treated with concentrations of 7, 15 or 30 μg/mL/kg FNZ, which suggest that FNZ did not affect the cell division index.Moreover, in the case of an acute treatment with the extrapolated doses commonly consumed by humans (0.5, 1.0 and 2.0 mg), the absence of toxicity of FNZ is noticeable.However, studies that have used high doses of FNZ have shown divergent results.One example is Namera et al. (2012), who showed that FNZ has acute toxic effects in high doses because FNZ caused the death of a woman who ingested high doses of FNZ and triazolam (another anxiolytic and sedative).
The evaluation of the aneugenic and clastogenic potential of FNZ, which was performed by the in vitro micronucleus test in HTC cells (Figure 3) and by the in vivo chromosomal aberration test in the bone marrow cells of Wistar rats (Figure 2), showed that the numbers of micronuclei and chromosomal aberrations were not statistically different from the negative control at all concentrations evaluated.These data suggest that FNZ does not possess aneugenic or clastogenic activity.Furthermore, the percentage of chromosomal aberrations did not differ according to sex, indicating that mutagenic activity does not occur in both males and females.
In this study, FNZ treatment did not result in mutagenicity; these findings are similar to those results obtained by Degraeve et al. (1985), who investigated the clastogenicity of this medicine by the chromosomal aberration test in the bone marrow cells of male mice intraperitoneally treated with 850 μg/kg for 12 to 72 hours.In addition, other studies have indicated that FNZ was not able to reversely mutate the Salmonella typhimurium TA100 strain when treated with a concentration of 5,000 μg/plate and did not cause the formation of mutations in the l5178Y gene in mouse lymphoma cells (Brambilla, Carrozzino, Martelli, 2007;Brambilla, Martelli, 2009).Additionally, FNZ treatment did not damage the singlestranded DNA of rat liver cells in vivo (Carlo et al., 1989) and did not cause chromosomal aberrations in the bone marrow cells of mice in vivo (Brambilla, Carrozzino, Martelli, 2007;Brambilla, Martelli, 2009).Although the results of this study have not shown any aneugenic, clastogenic or cytotoxic effects of FNZ, subchronic studies with FNZ should be performed to evaluate the long-term effects of daily consumption, which is typical in humans, and of higher doses to evaluate the effects of acute high concentrations, such as in situations of abuse.

FIGURE 2 -
FIGURE 2 -Percentage of the mitotic index and chromosomal aberrations for the negative (CO -) and positive (CO+) control groups and for groups treated with concentrations of 7 [1], 15 [2] or 30 [3] μg/mL/kg of flunitrazepam in vivo.*Statistically significant result compared with the negative control (p < 0.001).

TABLE I -
The mean cytokinesis block proliferation index (CBPI) of 1,500 control cells compared with cells that were treated with flunitrazepam