Download Secretion of hydrolytic enzymes by fungi strains isolated from

Secretion of hydrolytic enzymes by fungi strains isolated from patients before, during
and after radiotherapy
Introduction
One of the method which is used for cancer therapy is radiotherapy, which in
cases of head and neck tumors allows to reach similar effects as surgery and in
advanced cases is an element of therapy associate with other methods; it is used in
almost 60% of patients with malignant tumors and it gives 75-90% of cure (OsuchWójcikiewicz i wsp., 2010). Radiotherapy can cause on a great area of oral, pharynx
or larynx mucosa injure – epitheliolisis with acute post-radiation reaction of mucosa,
with deterioration of quality of life, disturbances of chewing and swallowing, also with
xerostomia. It predisposing to mechanical injures and development of infections, also
caused by fungi [1-8].
Aim
The aim of paper was mycological examination of patients with oral cavity,
pharynx or larynx carcinoma before, in 3 week, in last day and 3 weeks
after
radiotherapy and the estimation of release by fungi a hydrolytic enzymes.
Material and methods
43 patients (11 women and 32 men) in age between 45 and 85 (mean 63.09;
SD = 9.46) with oral cavity, pharynx or larynx carcinoma before, in 3 week, in last day
and 3 weeks after radiotherapy were examined. Among more than 2/3 (68.2%) had
fungi in first, 4/5 (80%) in second, about 3/5 (57.1%) in third and all (100%) in last
examination.
The mycological examination was conducted basing on procedure introduced
In Department of Biology and Medical Parasitology Medical University of Lodz [9].
Activity of hydrolytic enzymes was evaluated by using bioMerieux API ZYM test,
which is a semiquantitative method of determining 19 hydrolytic enzymes, which are
gathered in Table I. The results were read according to the instruction provided by
the producer. The activity of enzymes was expressed in nanomols of hydrolysed
substrate – according to the intensity of colour reaction in 5 scale: 0 – no reaction; 1
– 5 nanomols; 2 – 10 nanomols; 3 – 20 nanomols; 4 – 30 nanomols; 5 – 40
nanomols and more. Biotyping of strains was done according to Kurnatowska
classification [10], which is presented In Table II.
For statistical analyses prevalence and correlation with using Rho-Spearman
method were used. All calculations were done using STATISTICA v.7.1.
Results
More than 2/3 (68.2%) patients had fungi in the first, 4/5 (80%) in the second,
about 3/5 (57.1%) in the third and all (100%) in the last examination.
The releasing of enzymes differ; in I study α chymotrypsin
and
α
mannosidase, in II – β glucuronidase and α mannosidase, in III - α chymotrypsin,
and in IV – trypsin, α chymotrypsin, α galaktosidase and α fucosidase were not
active. The percentage of strains showing enzymes activity measured in nanomoles
of hydrolysed substrate are presented in Tables III-VI.
In first examination leucine arylamidas, esterase, valine arylamidase, acid
phosphatase and naphthol-AS-Bi-phosphohydrolase were the most active, much
more then alkaline phosphatase and N-acetyl-β -glucosaminidase (p < 0,05).
Detailed data are presented in Table III.
The analysis of hydrolytic enzymes activity of strains isolated during second
examination revealed that the highest activity had leucine arylamidase, acid
phosphatase, naphthol-AS-Bi-phosphohydrolase and β glucosidase; that activity was
higher than alkaline phosphatase, cystine arylamidase, β glucuronidase and α
mannosidase (differences statistically significant, p < 0,05). Detailed data are
presented in Table IV.
In third examination acid phosphatase, leucine arylamidase and naphthol-ASBi-phosphohydrolase were the highest, higher than lipase, cystine arylamidase,
tripsine, α chymotrypsine, α galactosidase, β glucuronidase oraz α mannosidase (p <
0,05). Table V include detailed date on this subject.
In 4th examination only leucine arylaminidase had the highest activity, which
was much higher then cysteine arylamidase and α mannosidase; details are in Table
VI.
The comparison of enzymes activity estimated during all examinations
revealed that the differences were present only among 3 enzymes: alkaline
phosphates, esterase and α glucosidase. The activity of alkaline phosphatase was
statistically lower in IV than in III examination while activity of esterase and of α
glucosidase were statistically lower in IV than in I and II examination.
Using rho Spearman method the analysis of correlation between activity of
particular hydrolytic enzymes and Candida albicans (the most common species) has
been done. In Table VII (n – analysis is not possible to conduct because of small
dispersal of enzyme activity) results are presented. Analyzing the relationship during
the first examination it was found that for C. albicans the increase of cysteine
arylamidase and decrease of alkaline phosphatase and β galactosidase activity were
characteristic while in the second examination the increase of esterase lipase and Nacetyl-β-glucosamidase as well as the decrease of acid and alkaline phosphatase
were observed. However in third examination lower activity of β glucosidase was
found. On the other hand in the last examination the highest activity of esterase,
cystine arylamidase and α mannosidase were demonstrated.
Taking into account the ability of fungal strains to release selected hydrolytic
enzymes 4 biotypes characteristic for particular species of Candida were determined.
Most strains were included the biotypes B, and C - at 31%, slightly less for D - 23%,
15% - were classified as biotype A. This data illustrates the Figure 1
Considering the different biotypes of the various species of Candida it was
found that the greatest diversity (classification in all four biotypes) was demonstrated
in the case of C.albicans and C.glabrata, the first of them classified mainly to biotype
B, while the second - to biotype C. Interestingly, that C.krusei and C.lusitaniae it was
observed a similar percentage of classification into three biotypes, in the first case to
biotypes A, B and C, while in the second - the A, B and D. It was also found cases of
the three strains which were classified to only one biotype. Detailed information is
graphically presented in Figure 2.
After taking into account subcategories of analyzed biotypes it was found that
the most strains of fungi were classified into biotypes D3 (22,5%), C6 (17,5%) and A
(15%), while the least - to C3 (7,5%), B1 (7,5%) and B2 (1,3%). Precise information
regarding classification of the different strains of the biotypes is summarized in Table
VIII.
By examining the ability to produce hydrolytic enzymes by the fungi it was
found in this study that were differences in their activity. In the first examination
inactive were α chymotrypsin and α mannosidase, in the second - β glucuronidase
and α mannosidase, in the third - α chymotrypsin, while in IV - trypsin, α chymotrypsin, α-galactosidase and α fucosidase. Different was the activity of hydrolases in
each investigation; the highest activity in all study had leucine arylamidase, and in
studies I-III - acid phosphatase and naphthol-AS-BI-phosphohydrolase. Moreover,
strains isolated during the first study were characterized by high activity of esterase,
and valine arylamidase.
Pytko-Polończyk et al [11] studied the enzymatic activity of 63 C.albicans
strains isolated from patients undergoing radiotherapy for cancer of the oral cavity
and oropharynx. Activity of 14 enzymes was found; a high was for acid phosphatase,
leucine arylamidase and naphthol-AS-BI-phosphohydrolase, while the total lack of
activity was for trypsin, α chymotrypsin, α galactosidase, β glucuronidase and α
fucosidase.
Plomer-Niezgoda et al [12] compared the enzymatic activity of strains isolated
from patients with primary lymphoma of the skin and bullous diseases treated with
immunosuppressive drugs, and found that fungi produced 11 of 19 enzymes: alkaline
phosphatase, esterase, esterase lipase, leucine arylamidase, valine arylamidase,
cystine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, glucosidase,  -glucosidase, N-acetyl--glucosyloaminidase; , the authors found that
there were differences statistically significant (0.0004 <p> 0.0003) between the
production of valine arylamidase and N-acetyl- -glucosyloaminidase by the strains
isolated from patients in comparison with strains derived from healthy individuals.
Evaluating the enzymatic activity of strains of C. alhicans from oral cancer
patients, between 1999 and 2003, Lukaszuk et al [13] found no significant
differences in hydrolase activity of strains isolated during these periods, but in 1999
the highest activity of the enzymes were characterized by : leucine arylamidase,
esterase and cystine arylamidase, while in 2003 – leucine arylamidse, esterase
lipase and esterase.
The strains of fungi isolated in the course of this study included mostly to
biotypes D3 (22,5%), C6 (17,5%) and A (15%), while the least to C3 (7,5%), B1
(7,5%) and B2 (1,3%).
Pytko-Polończyk et al [11] classified C.albicans strains isolated from patients
treated with radiation therapy in half the cases (49.2%) into biotype A,
to
Kurnatowska’s classification [10], the other mainly to B4 (42.9%), and single C6
(3.17%) and B1 (1.59%).
The most strains obtained from patients with primary lymphoma of the skin,
and bullous diseases treated with immunosuppressive drugs isolated by PlomerNiezgoda et al [12] were classified into biotype B3, B1 and D3.
Krajewska-Kułak et al. [14] denote biotypes of C. albicans
isolated from
patients with the oral cavity cancer and the most frequently qualified them to biotype
B4 (30%), much less to biotype E (4.8%), according to Kurnatowska’s classification
[10].
Strains of C. albicans isolated by Brajer et al. [15] from the sputum of patients
with lung cancer most often were classified as C6 (36,4%), B3 (24,2%)
and C1
(24,2%), much less as A (3.03%), B4 (3,03%) and D4 (3,03%) biotypes according to
Kurnatowska’s classification [10].
Pytko-Polończyk et al. [11] determined the biotypes of C. albicans isolated
from oral cavity of patients with nasopharyngeal cancer who underwent radiation
therapy, most often biotypes A (49.2%) and B4 (42.9%) were determined, rare
biotypes were: C6 (3,17%) and B1 (1,59%) according to Kurnatowska’s classification
[10].
Nowak [16] found that for C. albicans, C. glabrata and C. parapsilosis, isolated
from patients with implanted voice prosthesis, more frequently occurred biotype C6,
while for C. tropicalis strains
the most frequent was biotype B4; among strains
isolated from patients without voice prosthesis more often observed was biotype B3,
rarely D3; strains of C. glabrata and C. parapsilosis were qualified to biotype D3.
From the above, the literature data suggests that Candida strains isolated
from patients are characterized mainly by A, B1, B3, B4, C1, C3, C6, D1, D3, D4, E
biotypes.