The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides

doi:10.1016/0165-7992(90)90153-B

Mutation Research Letters

Volume 245, Issue 4, December 1990, Pages 245-249

https://doi.org/10.1016/0165-7992(90)90153-B Get rights and content

Abstract

Ultra-vital staining with acridine orange (AO) is introduced into the micronucleus assay with mouse peripheral blood cells. Peripheral blood was stained vitally by dropping whole blood on an AO-coated slide and covering the sample with a coverslip. With this method, reticulocytes are identified easily by their red fluorescing reticulum structure. The distinction between young and mature erythrocytes was clearer and less subjective than the distinction between polychromatic and normochromatic erythrocytes by Giemsa staining or by conventional AO fluorescent staining. Although the induction of micronucleated peripheral reticulocytes (MNRETs) was delayed by about 12 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optimal sampling time. It is concluded that bone marrow cells can be replaced by peripheral blood as material for the micronucleus assay.

References (7)

M. Hayashi et al.
An application of Acridine Orange fluorescent staining to the micronucleus test
Mutation Res.
(1983)
J.T. MacGregor et al.
A simple fluorescent staining procedure for micronuclei and RNA in erythrocytes using Hoechst 33258 and pyronine Y
Mutation Res.
(1983)
I. Amaki et al.
Fluorescent staining
Jap. J. Clin. Path.
(1978)

There are more references available in the full text version of this article.

Cited by (458)

Absence of adverse effects of Blutaparon portulacoides (A.St.-Hil.) Mears in mice exposed during pregnancy
2024, South African Journal of Botany
Blutaparon portulacoides (A.St.-Hil.), also known as capotirguá, pirrixu, and bredo-de-praia, is a bactericidal and anti-inflammatory agent used in folk medicine to treat leukorrhea. The present study evaluated the effects of an ethanolic extract of B. portulacoides (EEBp) on the reproductive performance, embryo-fetal development, and chromosomal stability of mice of pregnant female mice treated during the gestational period with oral EEBp (500 or 1000 mg/kg) or vehicle (1% Tween 80). EEBp did not alter biometric parameters, placental index, placental efficiency, or other parameters related to reproductive performance, neither embryo-fetal development and DNA integrity, but 500 and 1000 mg/kg EEBp decreased fetal and placental weight, respectively. Although the frequency of external and skeletal malformations did not differ between these groups and the control group, EEBp increased the frequency of reduced ossification of the presphenoid and xiphoid, which can be considered variants of normality. These findings indicate that EEBp is a safe and cost-effective treatment for leukorrhea during pregnancy, although caution should be exercised in its use.
Cytotoxicity, oral toxicity, genotoxicity, and mutagenicity evaluation of essential oil from Psidium glaziovianum Kiaersk leaves
2023, Journal of Ethnopharmacology
Members of the Psidium genus have been suggested in ethnobotanical research for the treatment of various human diseases, and some studies have already proven their popular uses through research, such as Psidium glaziovianum, which is found in Brazil's northeast and southeast regions and has antinociceptive and anti-inflammatory properties; however, the safety of use has not yet been evaluated.
This study investigated the safety of using essential oil obtained from P. glaziovianum leaves (PgEO) in vitro and in vivo models.
Cytotoxicity was evaluated in murine erythrocytes, while acute toxicity, genotoxicity (comet assay) and mutagenicity (micronucleus test) studies were performed using Swiss albino mice.
In the cytotoxicity assay, the hemolysis rate indicated a low capacity of PgEO to cause cell lysis (0.33–1.78%). In the acute oral toxicity study, animals treated with up to up to 5000 mg/kg body weight did not observe mortality or physiological changes. Neither dosage caused behavioral problems or death in mice over 14 days. The control and 2,000 mg/kg groups had higher feed intake and body weight than the 5,000 mg/kg PgEO group. Erythrocyte count, hemoglobin level, mean corpuscular volume, and MCV decreased, but serum alanine and aspartate aminotransferases increased. In the genotoxic evaluation, 5000 mg/kg PgEO enhanced nucleated blood cell DI and DF.
The present study describes that PgEO can be considered well tolerated in acute exposure at doses up to 2000 mg/kg, however the dose of 5000 mg/kg of PgEO should be used with caution.
Evaluation of the safety of ethanolic extract from Piper amalago L. (Piperaceae) leaves in vivo: Subacute toxicity and genotoxicity studies
2022, Regulatory Toxicology and Pharmacology
Piper amalago L. (Piperaceae) is traditionally used due to its anti-inflammatory, analgesic, diuretic, and antiparasitic properties. However, few studies have focused on its adverse effects, compromising its safe use. This study evaluated the toxicological safety of ethanolic extract from Piper amalago leaves (EEPA), through subacute toxicity and genotoxicity assays in rodents. In subacute toxicity, 100, 200 or 300 mg/kg of EEPA were tested in female Wistar rats, by gavage, for 28 days. For genotoxicity test, female Swiss mice were orally treated with 17.5, 175 or 1750 mg/kg of EEPA and the comet, micronucleus, and splenic phagocytic assays were evaluated. In subacute toxicity, the extract induced an increase in the food and water intakes, as well as in the liver absolute weight, and in the heart and kidney relative weights. EEPA also provoked alterations in histopathological analysis of liver and in hemato-biochemical parameters, evidenced by a decrease in hematocrit levels and albumin levels, and an increase in the number of platelets and in alkaline phosphatase and cholesterol levels. However, EEPA did not presented genotoxic nor mutagenic properties. EEPA showed hemato-biochemical toxicity profile in rats and should be used with caution, especially when for prolonged period.
Flavonoid-rich fraction of Croton blanchetianus Baill. (Euphorbiaceae) leaves: Chemical profile, acute and subacute toxicities, genotoxicity and antioxidant potential
2022, South African Journal of Botany
Croton blanchetianus Baill, commonly known as “marmeleiro”, is native to the Brazilian Caatinga is traditional used to treat joint pain and gastrointestinal disorders. The aim of the study was identify the chemical constituents by HPLC-DAD and determine the antioxidant activity of the spray-dried extract (SDE) aqueous (AqF) and ethyl acetate (EAF) fractions obtained from the leaves of C. blanchetianus; and to determine the acute (single dose, 2000 or 5000 mg/kg, p.o) and subacute (repeated dose, 250, 500 and 1000 mg/kg, p.o) toxicities, and genotoxicity (1000 and 2000 mg/kg, p.o) of the EAF in mice. In the acute oral toxicity assay the LD₅₀ was determined as > 5000 mg/kg. In the subacute toxicity, changes in weight gain, food and water intake, hematological, organ coefficients, oxidative stress parameters were not observed; however, changes in transaminases and leukocyte infiltration in the liver were observed at the highest dose. The genotoxicity test showed that high concentrations of EAF were not genotoxic in micronucleus and comet assays. The enrichment process was confirmed by HPLC and five flavonoids were identified. Our results provide evidence that the EAF was safe to use, although a long-term dose of 1000 mg/kg in should be avoided.
Biological safety of Syagrus coronata (Mart.) Becc. Fixed oil: Cytotoxicity, acute oral toxicity, and genotoxicity studies
2021, Journal of Ethnopharmacology
Syagrus coronata, popularly known as licuri, is a palm native to caatingas. The fixed oil extract of licuri nuts is used by the population of Northeast Brazil for therapeutic purposes, including as an antifungal, anti-inflammatory, and a cicatrizant agent. However, there is no scientific information on the possible harmful health effects of the oil and hence its medicinal usability is unknown.
We aimed to analyze the biological safety and possible antioxidant activity of fixed S. Coronata oil.
Chemical analysis of the oil was performed using gas chromatography with flame ionization detection (CG-FID). The cytotoxicity of varying concentrations of the oil (12.5, 25, 50, 100, and 200 μg/mL) was evaluated using the tetrazolium reduction assay in three cell lines: HEK-293 kidney embryonic cells, J774.A1 macrophages, and the tumor line Sarcoma-180 (S-180). Oral toxicity, genotoxicity, and mutagenicity tests were performed in mice which were administered a single dose of 2000 mg/kg of fixed licuri oil, by gavage. For acute toxicity tests, changes in blood and biochemical parameters, behavior, and weight were analyzed; histomorphometric analyses of the liver, kidney, and spleen were also performed. The comet assay and micronucleus (MN) test were performed to analyze genotoxicity. The antioxidant potential was assessed by the total antioxidant capacity (AAT) and DPPH elimination activity.
Licuri oil consists predominantly of saturated fatty acids, and lauric acid is the major compound. The highest concentrations of the oil showed low levels of cytotoxicity; however, LC50 was not reached in any of the tests. The acute toxicity study did not reveal any evidence of adverse effects in animals treated with oil; biochemical investigation of blood showed a decrease in blood concentration of total proteins and uric acid. The kidneys, spleen, and liver showed no morphological changes indicative of a pathological process. Genotoxic or mutagenic activity was not detected through both the comet assay and MN test. In addition, the oil showed low antioxidant activity in both methods.
Licuri oil from the stem of S. coronata did not present significant toxic effects as well as absence of genetic damage when administered orally. Future studies are needed to investigate its pharmacological potential.
Aqueous extract from leaves of Doliocarpus dentatus (Aubl.) Standl. relieves pain without genotoxicity activity
2021, Journal of Ethnopharmacology
Citation Excerpt :
In total, 200 cells/animal were visualized, and DNA fragment migration was determined according to comet class (Kobayashi, 1995; Oliveira et al., 2015). Two thousand cells/animal were analyzed under a fluorescence microscope (BIOVAL®, L Model, 2000A) with a 420–490 nm excitation filter and 520 nm barrier filter (Hayashi et al., 1990; Carvalho et al., 2015). Epifluorescence microscopy analyses of 200 cells per animal were conducted as described by Carvalho et al. (2015) using a fluorescence microscope with a 40 × objective, 420–490 nm excitation filter and 520 nm barrier filter (Bioval®, Model L 2000A, São Paulo, Brazil).
In the State of Mato Grosso do Sul, the watery sap of Doliocarpus dentatus is used to alleviate thirst, and the leaves of this species are used to relieve pain and swelling associated with inflammatory processes.
This study aimed to analyze the compounds of the leaves from the aqueous extract of D. dentatus (EADd) and evaluate its toxicogenetic and pain relief effects in animal models.
Compounds were identified in EADd by UHPLC-HRMS (Ultra high-performance liquid chromatography coupled to high resolution mass spectrometry). The oral dose of 17 mg/kg EADd, calculated according to ethnopharmacological uses, and doses between 30 and 300 mg/kg were used to test Swiss mice in formalin- and acetic acid-induced models of pain and behavior. EADd (100–2000 mg/kg) was assayed in mice by comet, micronucleus, and phagocytosis tests and by peripheral leukocyte counts.
Phenolic compounds and flavonoids as well as trigonelline and isoquercetin were identified in EADd. All oral doses of EADd exhibited antinociceptive activity, as indicated by a decrease in pain in both phases, a decrease in cold hypersensitivity induced by formalin, and a decrease in abdominal contortions induced by acetic acid. EADd did not alter the exploratory, motor or motivational activities of the animals. The comet and micronucleus tests indicated that EADd was not genotoxic and did not change the phagocytic activity or peripheral leukocyte count.
These results demonstrate that EADd could act as an antinociceptive agent that does not present genotoxicity. This study should contribute to justifying, in part, the popular use of D. dentatus in pain management, ensuring its safe use.

View all citing articles on Scopus

View full text

The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides

Abstract

Mutation Res.

Mutation Res.

Fluorescent staining

Jap. J. Clin. Path.