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2. Experimental
2.2.3. Conversion of testosterone by P. chrysogenum and
P. crustosum
The conversion of testosterone to its metabolites was
determined by isolating and identifying the products by
using thin-layer chromatography (TLC) and melting point
determinations [9,10].
2.1. Culture media, radioactive material, and chemicals
All culture media were prepared following Pitt’s procedures
[8]. The materials were obtained from Baker/or Bioxon (Bec-
ton Dickinson). Tritiated testosterone (1,2,6,7 [3H]T), specific
activity: 85–105 Ci/mmol, was obtained from New England
Nuclear (Boston, MA, USA), and radioinert testosterone (T)
was kindly provided by Syntex (Cuernavaca, Mexico). 5␣-
dihydrotestosterone, dihydrotestosterone (DHT), 3ϰ-hydroxy-
5-androstan-17-one, 3ϰ-hydroy-5ϰ-androstan-17-one, 4-an-
drostene-3,17-dione, and 5ϰ-androstane-3,17-dione, and
nicotinamide adenine dinucleotide (NAD) were acquired from
both Steraloids (Wilton, NH, USA) and Sigma (St. Louis, MO,
USA). Dextrose was purchased from Merck, and all solvents
were reagent grade, Baker.
2.2.3.1. Incubation. Two Erlenmeyer flasks containing 400
ml of sterilized potato dextrose broth [2,3] were inoculated
with P. chrysogenum or P. crustosum and stoppered with
foam plugs. The inoculated flasks were placed in a water
bath at 25°C and maintained under constant shaking for
24 h. Then 2 ml of a solution of testosterone at 1.3% in 95%
ethanol was added to the medium; the flasks were kept
under incubation conditions for 96 h longer. A third Erlen-
meyer flask was prepared as a noninoculated control.
2.2.3.2. Extractions. The cultures were extracted with 400
ml of dichloromethane saturated with water (3ϫ). The sol-
vent was evaporated to dryness in a rotary evaporator (Caf-
ramo vv 2000, Equipar, Mexico D.F.), and the extracts were
redissolved in 2 ml of ethanol.
2.2. Experiment 1
2.2.1. Isolation, maintenance, and identification of P.
chrysogenum and P. crustosum
The fungi (P. chrysogenum and P. crustosum) were isolated
from corn tortillas in a solid culture medium of potato dextrose
agar, as described by Holland et al. [2,3]. Species identification
was performed following Pitt’s criteria [8] by using three
different culture media—Czapek Yeast Autolysate Agar
(CYA), Malt Extract Agar (MEA), and 25% Glycerol Nitrate
Agar (G25Y)—and three different culture temperatures—5,
25, and 37°C, for 7 days. One plate of each CYA, MEA, and
G25Y was incubated at 25°C, another plate of CYA was
incubated at 37°C and the last one at 5°C.
2.2.3.3. Isolation and identification. An aliquot of the ex-
tract was spotted on a TLC plate to isolate and identify the
formed products. The unknown samples were run together
with steroid carriers (T, DHT, 3ϰ-hydroxy-5-androstan-
17-one, 3ϰ-hydroy-5ϰ-androstan-17-one, 4-androstene-
3,17-dione, and 5ϰ-androstane-3,17-dione) with the use of
three different solvent systems: benzene:ethyl ether, 6:4;
chloroform:acetone, 9:1 and benzene:ethyl acetate, 2:1 (v/
v). After development, some of the products were detected
and identified under 254 nm of ultraviolet light. DHT was
detected on the same plate after spraying with phosphomo-
lybdic acid reagent (8% in methanol).
2.2.2. Identifying characteristics
The remainders of the samples were filtered though a
20-g silica gel column [2] by using benzene as the solvent.
The products were collected, each in 2 ml of volume to
obtain crystals of the formed products. Previously, a column
was prepared under the same conditions, but it filtering the
carriers (T and DHT). To each of the 2-ml fractions was
added 0.5 ml of phosphomolybdic acid reagent (8% in
methanol), and each was heated at 70°C until a blue color
appeared in the presence of the steroids.
2.2.2.1. Growth data. Whether the isolate grows or not in
the different culture media and temperatures.
2.2.2.2. Macroscopic characters. In all three media, colony
measurements, conidial color, presence of exuded and sol-
uble pigments, reverse coloration, mycelia growth and col-
ony texture, medium buckling, and zonation were included.
2.2.2.3. Microscopic characters. The structure of the Peni-
cillus included whether the conidiophores were subsurface,
surface, or aerial; whether they form funicles; fascicles or
coremia length and wall; and the appearance of the stipes.
For Penicilli, the number of verticilli, vesiculated or not,
were examined. The number, length, location, and divergent
or appressed wall appearance of the rami was examined.
The number, length, wall appearance, form, and whether
divergent or appressed of the ramuli, metulae; ampulliform,
acerose, cylindrical or other form, number, length, and form
of the collula of the ramuli phyalides; and the form, chains,
size, and wall ornamentation of the conidia was determined.
2.3. Experiment 2
2.3.1. Uptake and conversion of [3H]T by P. crustosum
To verify the 5␣-reduction of T in the first experiment,
and to determine the uptake of [3H]T and its conversion to
DHT inside the mycelium of P. crustosum, cultures con-
taining [3H]T (47 M) and NAD (1 mM) were prepared in
25 ml of aqueous potato dextrose broth [2,3] at different pH.
These media were inoculated with fungus and maintained at
25°C in Erlenmeyer flasks for 5 days. The mycelia of the
fungi were separated by filtration through a mesh, washed,