Environmental and Molecular Mutagenesis 26:338-344 (1 995)

Genotoxicity of the Natural Cercaricides “Sucupira“ Oil and Eremanthine in Mammalian Cells In Vitro and In Vivo

Francisca da Luz Dias, Catarina Satie Takahashi, Elza Tiemi Sakamoto-Hojo, Walter Vichnewski, and Silvio Jose Sarti

Faculty of Medicine of Ribeirdo Preto, (F.d.l.D., C.S. J., E. J.S.-H.), Faculty of Philosophy, Sciences and letters of Ribeirdo Preto (C.S. J., E. J.S.-H.),

Faculty of Pharmaceutical Sciences of Ribeireo Preto (W. V., S.J.S.), Scio Paul0 University, Scio Paulo, Brazil

”Sucupira” oil and the lactone eremanthine, ex- tracted from Pterodon pubescens and Eremanthus elaeagnus, respectively, are known for their cercari- cidal action in experimental animals. Because of their biological effect, they have the potential to be used for the prophylaxis of schistosomiasis caused by Schistosoma mansoni. To test the clastogenicity of these agents, “sucupira” oil, either pure or di- luted in corn oil, was tested in vivo on Wistar rat bone marrow cells following dermal application. Metaphase analysis showed that the compound did not induce a significant increase in the frequencies of chromosomal aberrations. When eremanthine

was tested on BALB/c mice following gavage at doses of 100, 200, and 300 mg/kg bw, it did not induce structural or numerical chromosomal aberra- tions. In the in vitro treatment of human lymphocyte cultures, eremanthine also did not cause any in- crease in chromosomal aberrations or sister chra- matid exchanges at the following concentrations in culture medium: 1.25,2.50, and 5.00 kg/ml. From these results, under our experimental conditions, neither “sucupira” oil nor eremanthine showed clastogenic effects on mammalian cells in vivo or in vitro. 8 1995 Wiley-Liss, Inc.

Key words: “sucupira” oil, eremanthine, chromosomal aberrations, sister chromatid exchange, human lymphocytes

INTRODUCTION

Many plant species synthesize toxic chemicals, appar- ently as a primary defense against the hordes of bacterial, fungal, insect, and other predators. The variety of these toxic chemicals is so wide that phytochemists have been characterizing them for years, and new natural chemicals are still being discovered [Ames, 19831. Despite the muta- genic, teratogenic, and carcinogenic properties of plant- derived compounds [Ames, 19831, few toxicological stud- ies have been reported [Konstantopoulou et al., 19921. Among these compounds, some essential oils possess me- dicinal properties, and many of them are capable of pro- tecting experimental animals against infection by cercar- iae of Schistosoma mansoni [Pellegrino, 1967; Santos Filho et al., 19721. In many cases, active compounds have been shown to be sesqui- and diterpenes [Gilbert et al., 1 970al.

The oil extracted from the fruits of Pterodon pubescens Benth (“sucupira” oil) can prevent the penetration of the schistosome cercariae through the animal skin [Fascio et al., 19761. The major components of “sucupira” oil with cercaricidal activity are the diterpenoids 14,15-epoxyger- anylgeraniol and 14,15-dihydroxy- 14,15-dihydroxygera- nylgeraniol [Santos Filho et al., 1972; Fascio et al., 19761. Extracts from Eremanthus species and other Compositae

have demonstrated schistosomicidal properties. These species have been shown to contain a$-unsaturated y- lactones, which are responsible for their biological activ- ity [Vichnewski and Gilbert, 19721. The active component of the wood oil of Eremanthus elaeagnus Sch.-Bip. is the sesquiterpene lactone eremanthine [Garcia et al., 1976; Vichnewski et al., 1977; Herz et al., 19801. Many sesqui- terpene lactones are known for their cytotoxic and tumor- inhibiting properties. Toxic sesquiterpene lactones gener- ally contain one or more functional alkylating groups, most commonly an a$-unsaturated cyclopentanone or an a-methylene y-lactone group. The presence of these alkylating groups suggests the possibility of mutagenic and carcinogenic activity [Manners et al., 1978; Wall et al., 1988; Cassady et al., 19901. Because of their cercarici- dal properties, “sucupira” oil and eremanthine may af- ford protection against infection by Schistosoma mansoni, thus representing potentially useful compounds for the prophylaxis of schistosomiasis. On this basis, it is im-

Received April 24, 1995; revised and accepted August 10, 1995.

Address reprint requests to Dr. Francisca da Luz Dias, Faculdade de Medicina de Ribeirio Preto-USP, Avenida dos Bandeirantes 3900. 14049-900 Ribeirio PretolSio Paulo. Brazil.

0 1995 Wiley-Liss, Inc.

Genotoxicity of Natural Cercaricides 339

injected (ip) with 0.5 ml of 0.16% colchicine solution 90 min before death. One hundred metaphases per animal were analyzed per treatment.

Thirty-six BALB/c mice weighing approximately 30 g were divided into groups of three males and three females each and were treated by gavage with 0.3 ml of eremanthine diluted in a powdered milk solution at doses of 100, 200, and 300 mgkg bw. The animals were killed 24 hr after treatment, and chromosomal preparations from bone marrow were obtained by the technique of Ford and Hamerton [1956], adapted for mice by Rabello-Gay [1991]. The animals were injected with 0.3 ml of 1 % colchicine 2 hr before death. One hundred metaphases per animal were analyzed to determine the frequencies of chromosomal aberrations. The mitotic index was also determined (number of dividing cells/2,000 cells analyzed/animal).

The Kruskal-Wallis test was used for statistical analysis [Hollander and Wolfe, 19731. Fig. 1.

0 Structural formula of eremanthine.

portant to evaluate their possible clastogenic and cyto- toxic effects. The objective of the present study was to evaluate the ability of both “sucupira” oil and ereman- thine to induce chromosomal aberration and sister chro- matid exchanges (SCE) in mammalian cells in vivo and in vitro.

MATERIALS AND METHODS

Compounds

“Sucupira” oil and the sesquiterpene lactone eremanthine were ex- tracted from Pterodon pubescens Benth and Eremanthus elaeagnus Sch.-Bip., respectively. “Sucupira” oil was used either pure or diluted in corn oil, and eremanthine was diluted in dimethylsulfoxide (Me2SO- Merck) and milk powder at a concentration of 150 mgkg bw.

The molecular formula of eremanthine is C,,H,,C, and the spatial formula is shown in Figure I .

Bone Marrow Cells In Vivo

Wistar rats (Rartus norvegicus) and BALBk mice (Mus musculus) were obtained from the Animal Rearing Facility of the Faculty of Medi- cine of Ribeirb Preto (USP). Wistar rats weighing approximately 100 g were divided into groups each of two males and two females and submitted to two treatment schedules: (1) intraperitoneal (ip) injection with 0.1, 0.5, and 1 ml of pure oil and killed 24 hr after treatment or (2) dermal application of different doses for variable periods of time to the dorsum and tail.

Dorsum Treatment The interscapular region of the rats was shaved 1 day before topical

treatment with 0.5 ml of oil all day for 4 or 7 days, and the animals were killed 24 hr after the last day of treatment. The use of 0.5 ml of “sucupira” oil was based on U.S. Code of Federal Regulations (CFR), which considers this volume sufficient for toxic liquids to demonstrate an effect when administered to the skin [Emmett, 19911.

Tail Treatment The solution of “sucupira” oil and corn oil was applied to the tails

of the animals for 4 days at 5 and 50%. in addition to pure “sucupira” oil and corn oil. The animals were killed 24 hr after the last day of treatment.

Metaphase preparations were obtained from bone marrow cells by using the technique of Ford and Hamerton [1956]. The animals were

Cell and Culture Conditions

Human peripheral blood lymphocytes were obtained from two males and two females aged 20-30 years (non-smokers and in good health) and grown in 80% RPMI-1640 medium (Sigma) and 20% fetal calf serum (Cultilab) plus penicillin and streptomycin. Cells were stimulated with 4% phytohemagglutinin (prepared in the Genetics Department, Faculty of Medicine of Ribeir2o Preto). One milliliter of plasma was added to each 10 ml of culture medium, and the preparation was incu- bated at 37°C.

Preliminary tests were made with eremanthine to determine the con- centrations to be used on lymphocytes. More than 10.0 pg/ml totally inhibited cell division, so the concentrations tested were 1.25, 2.50, and 5.00 pg/ml. Distilled water, DMSO vehicle, and I-P-D-arabinofurano- sylcytosine (Ara-C) were used as the negative, solvent, and positive controls, respectively. The lymphocyte cultures were incubated and har- vested after 48 hr for chromosomal aberrations (CA) and after 72 hr for sister chromatid exchanges (SCE). 5-Bromo-2’-deoxyuridine (BrDU, Sigma) was added to the SCE cultures at a final concentration of 10 pg/ml. Mitotic arrest was achieved with colchicine 0.016 % (Sigma) 1:45 hr before fixation. After hypotonic treatment (0.075 M KCI for 10 min) and fixation (methano1:glacial acetic acid, 3: 1, three times), the cells were dropped on cold wet slides, air dried, and stained in Giemsa diluted in phosphate buffer ( 1 :30) for CAs and by the fluorescence plus Giemsa technique (FPG) of Perry and Wolff [I9741 and Korenberg and Friedlender [I9741 for SCEs. All slides were coded and 100 cells were analyzed from each culture for chromosomal aberrations, and 2,000 cells were scored for determination of the mitotic index (48-hr cultures). One hundred cells from each culture were scored for M 1, M2, and M3, and 50 second-division cells were scored for SCEs (72-hr cultures). The cell proliferation index (PI) was obtained by the following equation [Krishna et al. 19911:

PI = (IMI + 2M2 + 3M3)/100

Statistical analysis was carried out using the Friedman test [Hollander and Wolfe, 19731 to compare each treatment to the negative control cells. Data from the positive control cultures (Ara-C) were not included in the statistical analysis.

RESULTS

“Sucupira“ Oil Applied to Wistar Rat Bone Marrow Cells

The test with ‘‘sucupira” oil administered intraperito- neally to Wistar rats showed toxic effects at doses of 0.5 and 1.0 ml, which caused the death of 25 and 50% of the

340 Dias et al.

TABLE 1. Mitotic Index, Number of Chromosomal Aberrations in Bone Marrow Cells From Wistar Rats Treated i.p. With 0.1: 0.5 and 1.0 ml of “SucuDira” Oil for 24 h

Cells with Chromosomal aberrations Dose Metaphases (mVanimal1 (animal) MI (%I G’ G“ B’ B“ F’ P Total aberrations f % )

Control 400(4) 9.73 3 0 0 2 0 0 5 5( 1.25) 0.1 4W4) 3.70 2 0 2 0 1 1 6 6( 1 S O ) 0.5 300(3)* 2.90 0 4 2 0 0 0 6 6(2.00) 1 .o 200(2)** 3.20 I 0 1 0 0 0 2 2( 10.00)

* died one animal. **died two animals, G’ = Chromatid gap, G = Chromosome gap, B‘ = Chromatid break, f3“ = Chromosome break, F’ = Single fragment, P = Double fragment

TABLE II. Number of Chromosomal Aberrations in Bone Marrow Cells From Wistar Rats Treated Dermally (Dorsum) With 0.5 ml of “Sucupira” Oil for 4 and 7 Days and Dermally (Tail) With Different Concentrations of “Sucupira” Oil for 4 Days; 400 Metaphasemreatment Were Analyzed

Cells with Chromosomal Aberrations Concentration Fixation (treatment time) time (h) G’ B“ F’ R Dic Total aberrations (%)

Dermal treatment on the dorsum with 0.5 ml of “sucupira” oil for 4 and 7 days

corn oil (7 days) 48 7 4 I 0 0 12 12(3.00) 100% (4 days) 24 1 1 8 3 1 0 23 23t5.75) 100% (7 days) 24 15 5 0 0 1 21 21t5.25) 100% (7 days) 48 6 10 1 1 0 18 1 Q4.50)

Dermal Treatment on the tail with different concentrations of “sucupira” oil for 4 days

corn oil (4 days) 24 4 3 0 0 0 7 7( 1.75)

50% (4 days) 24 2 1 0 0 0 3 3(0.75) 100% (4 days) 24 1 1 0 0 0 2 2(0.50)

G‘ = Chromatid gap, B’ = Chromatid break, F’ = Single Fragment, Dic = Dicentric

5% (4 days) 24 1 3 0 0 0 4 4( 1 .00)

animals, respectively. A reduction in the mitotic index (IM) of the bone marrow cells was observed in the surviv- ing animals at all doses (Table I). This result led us to use the dermal route of administration that, in addition to being less toxic, is important because it is the route through which “sucupira” oil has shown efficiency in inhibiting cercarial penetration in rodents [Mors et al., 19671. The results of the experiments in which the animals were submitted to dermal treatment on the dorsum and tail with “sucupira” oil are presented in Table 11. The variations in frequency of aberrations in bone marrow cells between treated and control groups were not signifi- cant at the 5% level (P = 0.3670). The group of animals treated for 4 days and killed 24 hr later showed 5.75% higher frequencies of aberrations, but the difference was not statistically significant. When “sucupira” oil was emulsified in corn oil and applied dermally to the tail, the toxic effects observed (nose running, lack of appetite, and weight loss) in the experiment in which the oil was applied to the dorsum were not detected. The frequency of chromosome aberrations did not differ significantly between treated groups and the negative control (P = 0.289).

Eremanthine Applied to BALB/c Mouse Bone Marrow Cells

The 400-mg/kg concentration of eremanthine proved to be highly toxic as all animals had convulsions and were dead 22 hr after treatment by gavage. The concentra- tions of 100,200, and 300 mgkg had no cytotoxic effects as no significant changes in mitotic index were observed. With respect to chromosome aberrations, no differences were observed among animals or between treatments with different eremanthine concentrations and controls (P = 0.88). As expected, the powdered milk solution used as a solvent had no cytotoxic or clastogenic effects (Table III).

Eremanthine Applied to Human Lymphocytes

The distribution of the various types of chromosome aberrations obtained in human lymphocytes treated with eremanthine in vitro is presented in Table IV. Gaps were the most constant structural aberrations, and their fre- quency in treated cultures was statistically higher than in controls (P = 0.0388). However, when the total number of chromosome aberrations was considered, the differences

Genotoxicity of Natural Cercaricides 341

TABLE 111. Mitotic Index, Number of Chromosomal Aberrations and Aberrant Cells in Bone Marrow Cells From Balb/c Mice Treated by Gavage With Eremanthine: 600 Metaphaseflreatment

Cells with Chromosomal Aberrations Concentration (mglKg bw) MI (%) G‘ B’ F’ Total aberrations (%)

control Milk (150) Eremanthine 100 200 300 CP (8)

2.62 1 1 2 2.87 4 2 1

4 7

1.02 I 5 0 6 2.72 4 3 I 8 2.10 5 1 I 7 2.45 13 70 0 83

4(0.67) 7(1.17)

6( 1 .00) 8( I .33) 7( I . 17)

57(9.5)

G’ = Chromatid gap, B’ = Chromatid break, F’ = Single fragment, CP = Cyclophosphamide

TABLE IV. Mitotic Index, Chromosomal Aberrations and Cells With Aberrations in Human Lymphocyte Cultures Treated With Different Concentrations of Eremanthine: 400 MetaDhaseslTreatment

Cells with Chromosomal Aberrations Concentration pg/ml MI (%) G’ B B” F’ P Exc Total aberrations (%)

control 7.36 1 5 1 0 1 0 8 7( 1.75) DMSO(0. I %) 5.14 1 2 2 3 0 I 9 7( 1.75) Eremanthine 1.25 4.55 3* 10 0 0 3 2 18 I4( 3.50) 2.50 5.09 9* 4 1 0 0 0 14 14( 3.50) 5.00 5.23 4* 5 0 2 0 0 9 9(2.25) Ara-C(2.5 X lo-’)# 4.05 5 27 5 0 1 4 42 40( 10.00)

G‘ = Chromatid gap, B‘ = Chromatid break, B“ = Chromosome break, F‘ = Single fragment, P = Double fragment, Exc = Exchange, *Statistically significant (p < 0.05) (Friedman test), # Not included in the statistical analysis.

were not significant between the various treatment groups or among individuals. The mitotic index values demon- strate that eremanthine was not cytotoxic at the concentra- tions used. Eremanthine caused no increase in SCE fre- quency or changes in the cell proliferation index in lym- phocyte cultures when compared with the control cultures. Although Ara-C produces significant chromo- somal damage in lymphocytes cells, it has not been found to cause an increase in SCEs (Table IV).

DISCUSSION

The clastogenic activity of chemicals on somatic tissue in vivo is usually assessed by cytogenetic analyses in rodent bone marrow cells. The choice of this tissue is mainly due to the large number of easily available mitotic cells and to the thick network of blood vessels, which allows a good contact of the target cells with the adsorbed or administered compound [Barale et al., 19921. Based on the chemical properties and biological reactivities of “sucupira” oil, the dermal route of admistration was de- termined to be the most appropriate.

Dermal application of “sucupira” oil has been reported to have cercaricidal effects on mice [Mom et al., 1967; Santos Filho et al., 1972; Gilbert et al., 1970a,b]. In the

present assay, the animals were exposed to the oil at a subacute rate (4 and 7 days), which, according to Legator and Ward [1991], is the most appropriate for this type of treatment. Because “sucupira” oil is a polar compound, as are lipids, it has the ability to pass through the lower layers of the epidermis until it reaches the dermis, where it enters the systemic circulation [Klaassen and Rozman, 19911.

“Sucupira” oil had a toxic effect when applied to the dorsal region of the epidermis, a fact that was not ob- served when the oil was applied to the tail. These observa- tions lead us to assume that the dorsal region may be more permeable than the tail, thus permitting easier ab- sorption of the oil. According to Feldmann and Maibach [ 19691, the skin is the major point of entry of many agents, and the amount absorbed by the organism depends on the dose applied and on the region of the body that received the treatment. In the regions in which the epidermis layer is thinner, absorption is more rapid than in regions where the layer is thicker [Stoughton, 19891, as was observed in the present study. With respect to the frequency of chromosome aberrations, ‘‘sucupira” oil was not clasto- genic when administered dermally to the dorsum and tail. These results may have been due to the biotransformation of this oil in the liver, with inactivation of the agent, or rapid excretion of its metabolites before reaching the tar-

342 Dias et a[.

TABLE V. Frequencies of SCEs and Proliferation Index (PI) in Human Lymphocyte Cultures Treated With Eremanthine. Two Hundred Cells per Treatment Were Analyzed for SCEs and 400 Metaphases per Treatment Were Analyzed for PI Determination

Concentrations udml MEAN ?SEM Total MI M2 M3 PI

SCE

Control 9.67 1.49 1933 I 84 191 25 I .60 DMSO(O.I%) 9. I4 1.1 1 1838 138 207 55 1.77 Eremanthine I .25 9.01 I .65 1801 224 153 23 I .50 2.50 9.85 1.40 1969 240 131 29 I .47 5.00 10.76 1.38 2157 202 172 26 1.56 AraC(2.5 X lo-') 9.46 1.55 1892 23 1 144 25 1.58

MI = First mitosis, M2 = Second mitosis, M3 = Third mitosis, SEM = Standard error of the mean

get organ (bone marrow). Alternatively, the concentration of the oil or its possible active metabolite may not have been sufficient to induce changes in bone marrow cells. A third hypothesis is that the bone marrow may not be the target organ of this compound.

Because it is only soluble in DMSO, intraperitoneal administration of eremanthine requires a much greater volume of DMSO than is recommended by guidelines (0.1 ml) [Preston et al., 1987a1, so we opted for gavage, using powdered milk solution, which, besides not being toxic, was better accepted by the animals, assuring total drug ingestion. Milk was not cytotoxic or clastogenic (Table III), as observed by Tavares and Takahashi [ 19941, who used milk to dilute the alkaloid boldine. Eremanthine proved to be quite toxic at the dose of 400 mgkg bw, causing death for all animals 22 hr after treatment by gavage. Eremanthine may have a hepatotoxic effect, be- cause, according to Chapman et al. [1989], the toxicity of lactones to mice is probably a consequence of their reaction with the binding sites of the substrates of cyto- chrome P-450, forming metabolites that may contribute to the inhibition of the mixed-function oxidase system, which plays an important role in liver metabolism.

Despite its high toxicity at the dose of 400 mg, at lower doses there was no toxic effect. At doses of 100,200, and 300 mgkg bw, eremanthine did not inhibit cell division or have a clastogenic effect on mouse bone marrow cells. Similarly, the lactones erioflorin metachrylate, erioflorin acetate, and ovatifolin acetate also did not induce an in- creased number of micronuclei when tested on this same system [Cea et al., 19901. Similar observations have been made with the lactones eremantholide-C and 15-deoxy- goyazensolide [Vicentini-Dias, 19921, and goyazensolide [Mantovani et al., 19931 when tested in vivo on Wistar rat bone marrow cells. Eremanthine had no clastogenic effect on human peripheral blood lymphocytes treated in vitro. Although the frequency of gaps at concentrations of 1.25, 2.50, and 5.0 pg/ml was significantly increased when compared with the control group, the same was not

observed when the total number of aberrations (gaps and breaks) was considered (Table IV).

Gaps are considered by some investigators to be of debatable genetic significance because their presence does not always lead to chromosome aberrations in the subse- quent cell divisions [Preston et al., 1987b; Brusick, 19871. Electron microscopic studies have revealed that gaps do not appear to be a true discontinuity in the chromatid, because chromosomal material can be visualized between the proximal and distal regions of these lesions [Scheid and Traut, 1971; Brogger, 19821. However, several stud- ies on clastogenicity induced by chemical compounds have suggested that gaps may be associated with mutage- nicity [Goetz et al., 19751 and may have some biological meaning representing a toxicological phenomenon [Di Paolo and Popescu, 1976; Anderson and Richardson, 198 11. Galloway et al. [ 19861 included gaps in the general computation of aberrations induced by occupational expo- sure to ethylene oxide. Despite these contradictory opin- ions, we think that gaps should be considered because they increase the total number of chromosome aberrations and their presence is accompanied by other complex chro- mosome alterations more characteristic of clastogenicity.

When the groups treated with eremanthine were com- pared with their controls, no increase in SCE frequency or changes in mitotic index and cell proliferation index were observed (Table V). This suggests that, under the conditions of the assay, eremanthine was not involved in the processes of cell division. Another important observa- tion is that, in assays carried out under the same experi- mental conditions as used here, the lactones eremanthol- ide-C and 15-deoxygoyazensolide [Vicentini-Dias, 19921 and the pseudoguaianolide goyazensolide [Mantovani et al., 19931 also induced no increase in the frequency of chromosome aberrations or SCEs in human lymphocyte systems in vitro.

Ara-C used as a positive control induced an evident clastogenic effect, with an increase in chromosome aber- rations (Table IV), without increasing the frequency of

Genotoxicity of Natural Cercaricides 343

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SCEs (Table V), under the same experimental conditions; perhaps this is due to the Ara-C dose used (2.5 X lo-’). There is considerable evidence that the mechanisms in- volved in the prodution of chromatid breaks are different from those causing SCEs [Benedict and Jones, 19791. It has also been shown that, although X-irradiation induces a marked increase in chromosomal aberrations, only a light increase in SCEs is produced [Peny and Evans, 19751.

Lactones with unsaturated bonds in their structure, as is the case for eremanthine, have the ability to react with the nucleophilic centers of cellular macromolecules [Jones et al., 1981; Woynarowski and Konopa, 19811. When this reaction occurs with the thiol group of the tripeptide glutathione, blockade of the activity of this compound may occur in relation to other macromolecules such as DNA [Anick et al., 1983; Woerdenbag et al., 1989a,b]. The mutagenic potential of lactones may not always be related to their structure, since the mutagenicity of himenovine was attributed to the uncommon molecule bishemiacetal rather than to the sulfhydryl group a-meth- ylene y-lactone [Manners et al., 19781. Lactones may also have antimutagenicity properties, as is the case for a- methylene-butyrolactone, 5,6-dihydro-2H-piran0-2,1 -es- culetin, and 4-methylesculetin, which inhibit the muta- tions induced by 4-nitroquinoline 1 -oxide (4NQO) and N-methy 1-N-nitro-N-nitrosoguanidine (MNNG) in Esche- richia coli strains [Kuroda et al., 19861.

Thus, the present data permit us to conclude that, under the experimental conditions employed here, ‘ ‘sucupira” oil had no clastogenic effect when tested in vivo and that eremanthine had no clastogenic or cytotoxic activity in vivo or in vitro.

ACKNOWLEDGMENTS

We thank Miss Sueli A. Neves and Mr. Luiz August0 da Costa Jr. for technical assistance. F.L.D. thanks the Department of Genetics, Federal University of Par& for support during her stay in Ribeirgo Preto, when the re- search was performed. This research was supported by Capes, CNPq, and FINEP.

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Accepted by- D.A. Casciano