The effects of triiodothyronine and thyroxine on metabolism and growth of bacteria were studied. It was observed that over a certain range of concentration thyroxine and triiodothyronine produced increase in 14CO2 release from [14C]-labeled glucose and also stimulated bacteria growth

Gaitonde, B. B.

Ganatra, R. D.

Nimbkar, Y. S.

Shanta, M. S.

Singh, K. T.

English

Publications of the IAS Fellows

jnm/ IN VITRO NUCLEAR MEDICINE
Thyroid Hormones And [14C] Glucose
Metabolism in Bacteria
K.T.Singh, R.D. Ganatra, M. S. Stianta, Y.S. Nimbkar, and B.B.Gaitonde
Bhabha Atomic Research Centre and Haffkine Institute, Parel, Bombay, India
The effects of Iriiodothyronine and thyroxine on metabolism and growth
of bacteria were studied. It was observed that over a certain range of con
centration thyroxine and triiodothyronine produced increase in 1'>COlre
lease from ["C] -labeled glucose and also stimulated bacteria growth.
J NucÃ-Med 18: 736-739, 1977
It has been well established that the thyroid hor
mones, acting through augmented transcription of
DNA, can affect protein synthesis in cell suspensions
and tissue cultures in vitro (1), but there are very
few reports about their effects on bacterial growth
and metabolism. Increase in glucose oxidation by
yeast cells in the presence of thyroid hormones has
THYROXINE TRIIODOTHYRONINE
002 ugm )
2 i
TIME IN HOURS
been reported (2,3). Biswas (4) in a brief letter to
the editor of Lancet has referred to the luxuriant
growth of E. coli cultures in the presence of thy
roxine.
We have studied the effects of thyroxine and tri
iodothyronine on release of 14CO2as an end-product
of bacterial metabolism of [14C]-labeled glucose.
During the course of this work, we also observed
that these hormones stimulate the growth of mi
crobes.
MATERIALS AND METHODS
Method for detection of 14CO2.The detecting de
vice consists of a liquid-scintillation vial lined with
Whatman No. 42 filter paper. The paper has pre
viously been soaked in scintillation fluid (PPO 10 g,
POPOP 0.125 g, dioxane 100 ml) and in NaOH and
then dried before installation in the vials. Each vial
contains within it another narrow cylindrical vial with
several holes at the upper end. The inner tube con
tains nutrient broth, [U-14C] glucose, and a Siaphy-
lococcus aureus culture. The vials are incubated in a
water bath at 37Â°C,shaken continuously to encour
age escape of 14CO2 from the aqueous phase. The
14CO2 evolved in the inner vial as a result of bac
terial metabolism is gradually trapped on the alkali-
impregnated filter paper lining the outer vial.
FIG. 1. Effect of varying concentrations (5-1000 ng/ml) of thy
roxine and triiodothyronine on release of "CO2 from a culture of
S- aureus containing [I4C] glucose.
Received Nov. 23, 1976; revision accepted Feb. 1, 1977.
For reprints contact: R. D. Ganatra, Radiation Medicine
Centre, Bhabha Atomic Research Centre, c/o Tata Memorial
Hospital, Parel, Bombay 400 012, India.
736 JOURNAL OF NUCLEAR MEDICINE
IN VITRO NUCLEAR MEDICINE
TABLECone,
of
thyroxine(M9)00.040.11.02.01.
EFFECT OFTHYROXINE ON "CO, RELEASE FROM S. AUREUS IN SEVENXP RIMENTS"COa
cpm aÂ»3hr9791388923139432410530323127417045932633884247912435436326416926541413476746381411622454076343323809777272513501146721377741
TABLE 2. EFFECTIVE DOSE OFTHYROXINEFOROrganismS.
aureiÂ»E.
coliS.
typ/iiS.
dysenterioeC.
welchiiP.
YulgarisM.
tuberculosisVARIOUSThyroxine(ug/ml0.040.10.10.10.11.010.0ORGANISMSCpm
withcpm
in6,
8,8,1663910610thyroxinecontrol8,7, 7, 9
The counting was done in a liquid scintillation
counter. Since the vials were not opened throughout
the incubation period, it was possible to obtain cumu
lative measurements of the evolved 14CO2by peri
odic counting. A similar system was described orig
inally by Buddemeyer (5), but we have modified his
technique to some extent to suit our needs.
Effect of triiodothyronine and thyroxine on "t'O^
release. Experiments were performed with concen
trations of L-thyroxine ranging from 5-1000 ng/ml.
The inner vial held 1-2 ml nutrient broth containing
2 /Â¿Ci[U-14C] glucose and 0.1 ml of an innoculum
containing 10 million cells from an overnight culture
of S. aureus. Vials were kept in the shaking water
bath at 37Â°C,and the 14CO2was counted in liquid
scintillation counter at various time intervals. Simi
lar experiments were carried out with triiodothy
ronine in concentrations ranging from 5-1000 ng/ml.
In similar experiments we also studied the effects
of various concentrations of thyroxine on CO2 pro
duction from glucose in other organisms, e.g., E. coli,
S. typhi, C. welchii, S. dysenteriae, P. vulgaris, and
M. tuberculosis.
Effect of triiodothyronine and thyroxine on bac
terial growth. The control culture and the culture
containing thyroxine were plated on nutrient agar
plates after 14CO2counting as described above con
tinued up to 4 hr. The plates were then incubated
at 37Â°Cfor 24 hr.
Incorporation of tritiated thymidinc in the pres
ence of triiodothyronine and thyroxine. A 5. aureus
culture (10 million cells in 2 ml nutrient broth) was
incubated at 37 Â°Cfor 24 hr with 10 ,*Ci of [3H]
thymidine (sp. act. 10,400 mCi/m M) with and
without thyroid hormones. At 24 hr the cultures were
centrifuged and the cells washed three times with
normal saline and then digested for 15 hr in 1 ml
hyamine hydroxide. One-tenth ml of hyamine was
counted in 15 ml scintillation fluid (6,7).
RESULTS
The effects of thyroxine and triiodothyronine on
release of 14CO2are shown in Fig. 1. Thyroxine at
5-20 ng/ml had no effect. With increased concen
tration to 40-100 ng/ml, there was an eightfold in
crease in counts within 1 hr, but 1.0 jug/ml of thy
roxine actually decreased 14CO2 release. What was
most striking was the very narrow range for the effec
tive concentration of thyroxine. Triiodothyronine
produced the same effects, but somewhat earlier and
at lower concentrations. The experiments were re
peated seven times and same pattern of 14CO2 re
lease could be observed every time. The results are
given in Table 1.
The experiments were carried out with other or
ganisms (Table 2) and showed that the concentra
tion of thyroxine causing maximum stimulation of
14CO2release varied from 40-1000 ng/ml, but the
pattern of 14CO2release was almost the same with
each type of organism.
Besides the 14CO2 release, it was observed that
growth in the culture containing thyroxine was far
more abundant than the control cultures, and the
data indicating growth paralleled those measuring
14CO2evolution. Up to a point growth was stimulated
by increasing amounts of thyroxine, but higher thy
roxine concentrations inhibited the growth (Fig. 2).
Table 3 shows that the growth of S. aureus, as
measured by incorporated tritiated thymidine, is in
fluenced by thyroid hormone in a similar manner.
Bacterial production of 14CO2 from labeled glu
cose has been used to monitor bacterial contamina
tion in various pharmaceutical products and in blood
Volume 18, Number 7 737
SINGH, GANATRA, SHANTA, NIMBKAR, AND GAITONDE
FIG. 2. Effect of thyroxine on growth of a S. Ã¡ureas culture.
From left, first nutrient-agar slant is plated from a control culture
without thyroxine, second and third from cultures containing 40 and
100 ng thyroxine, respectively, the last plated from a 1-/^g/ml cul
ture. Experiments performed with triiodothyronine gave similar
results.
cultures (8). This in vitro radiorespirometric method
reduces time for a sterility test to 8â€”10hr, compared
with the 8-10 days required by the method in the
pharmacopeia. The technique is applied to antibiotic
sensitivity testing, where the results can be obtained
within 2 hr compared to 24 hr by standard meth
ods (9). The addition of thyroxine or triiodothy
ronine to the culture medium is likely to reduce the
14CO-jdetection time still further in the preceding
methods, since 14CO2is released much earlier from
cultures containing triiodothyronine or thyroxine.
Results of experiments carried out to show this
showed that a S. aureus culture containing 60 or
ganisms and 100 ng thyroxine gave 7 times the count
rate of the thyroxine-free control, and this reduced
the detection period from 4 hr to 0.5 hr (Table 4).
TABLE 3. EFFECT OF THYROID HORMONES
ON PHI THYMIDINE INCORPORATION IN
BACTERIA IN TWOEXPERIMENTSConcen
tration
ofT4(jug/ml)00.040.11Counts/min3219361017304357957163916276324081Cone,ofT (/ifl/ml)0 .020.040.11.0Counts/min3219125939171430046305716146791001357895761
TABLE 4. RELEASE OF "CO FROM A SMALL
NUMBER OF ORGANISMS (S. AUREUS) IN
PRESENCE OF THYROXINE IN TWOEXPERIMENTSCounts/minNo
oforganisms60ConditionWithoutthyroxineWiththyroxine0.5
hr112179882hr1340941244hr7016018538424hr860215029308185
DISCUSSION
We have observed that both triiodothyronine and
thyroxine have a stimulatory effect on 14CO2release
as well as growth of bacteria over a certain range of
concentrations, while above that range it has an
inhibitory effect. The optimum concentration varies
with the type of bacterium. As in other in vitro sys
tems, we could also observe that triiodothyronine
acts much earlier and at lower concentration. It was
surprising to observe that the relative effectiveness of
thyroxine compared with triiodothyronine was of
the same order of magnitude in microbes and in man.
Iodide in the same concentration as thyroid hor
mones had no effect on growth of organisms. The
culture medium used in our experiments contained
50-80 /xgm/ml of iodide, and the addition of a frac
tion of a microgram was highly unlikely to produce
any significant difference.
It would be interesting to study the fate of radio
active triiodothyronine or thyroxine in the metab
olism of bacteria. Experiments of such a type are
under study.
Immediate practical application of these observa
tions would be in augmenting the sensitivity of in
vitro radiorespirometric studies for detection of con
tamination with microorganisms in pharmaceutical
and hospital laboratories.
The growth-promoting properties of thyroid hor
mones may also be useful in the culture of recal
citrant organisms like M. tuberculosis and M. leprae.
Probably of more fundamental importance is the
ubiquitous requirement of thyroid hormones by liv
ing cells and the roles these hormones may have in
the control and propagation of pathogens in man.
Lastly, can we speculate that the well-established
action of thyroid hormones on mitochondria is in a
way representative of its action on bacteria if one
believes that the mitochondrion is a symbiont bac
terium incorporated in the primitive- eukaryotic cell?
REFERENCES
1. TATA JR, WIDNELL CC: Ribonucleic acid synthesis dur-
738 JOURNAL OF NUCLEAR MEDICINE
IN VITRO NUCLEAR MEDICINE
ing the early action of thyroid hormones. Blochem 3 98: vitro studies of lymphocyte reactivity. Proc Soc Er p Biol
604-620, 1966 Med 140: 40-44, 1972
2. GuTEMSTEiN M, MARX W: Stimulation of yeast res- 7 KISSLING M, SPECK B: Simple micromethod for test-
piration by L-thyroxine. J Biol Chem 229: 599-602, 1957 ing phytohaemagglutinin response of lymphocytes. Lancet
3. WAINFAN E, MARX W: Effects of thyroxine and some j. 451.452 1972
related compounds on bacterial oxidations. J Biol Cliem214- 441-445 1955 8- DELAND FH, WAONER HN: Automated radiometrie
4. BISWAS SK: Effect of thyroxine on bacterial growth in detection of bacterial growth in blood cultures. J Lab Clin
vitro. Lance, 2: 716-717, 1975 Mfd â„¢ 529~5i4' 197Â°
5. BUDDEMEYEREU: Liquid scintillation vial for cumu- 9. DEBLANCHJ, CHARACHEP, WAGNERHN: Automatic
lative and continuous radiometrie measurement of in vitro radiometrie measurement of antibiotic effect on bac-
metabolism. Appi Microbiology 28: 2, 177-180, 1974 terial growth. Antimicrob Agents Chemother 2: 360-366,
6. PAULY JL, SOKALJE: A simplified technique for in 1972
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Thyroid hormones And [<SUP>14</SUP>C] glucose metabolism in bacteria

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Thyroid hormones And [<SUP>14</SUP>C] glucose metabolism in bacteria

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