The protocol for the bioassay with the mitochondria of beef heart frozen at -22 °C, (developed by Iero, Manente, Bragadin and Perin, in Chemosphere, 52, 2003) requires that the reaction cell is thermostatically controlled at 25 °C. This value was chosen because it is used as a reference for the state environmental standard (Standard Ambient Temperature and Pressure, SATP).
The choice is not, therefore, been supported by assessments on the effectiveness of the test at this temperature, but was dictated by the practice of reporting results to a standard temperature value. 
Finally, it was decided to make a comparison between the working temperature of 25 °C and the 37 °C one, chosen as close to cattle body temperature (estimated to be 38.6 °C), then "normal" working temperature for mitochondria extracted from heart

A. Iero

A. Zuin

G. Ravagnan

M. Bragadin

S. Manente

MANENTE, Sabrina

ZUIN, ALESSANDRA

BRAGADIN, Marcantonio

RAVAGNAN, Giampietro

Archivio istituzionale della ricerca - Università degli Studi di Venezia Ca' Foscari

test 25 °C 37 °C m R2 m R2 1 -0,30 0,967 -1,90 0,999 2 -0,30 0,976 -2,06 0,998 3 -0,37 0,991 -1,95 0,998 4 -0,32 0,992 -1,93 0,998 5 -0,32 0,985 -1,73 0,997 6 -0,26 0,954 -1,80 0,998 7 -0,42 0,987 -1,93 0,996 8 -0,36 0,896 -2,05 0,998 average -0,33 0,968 -1,91 0,998 FM22 end point                 DR = 1 – n/m  n = m , when testing substance(s) don’t show  acute toxicity for mitochondria  n < m , when testing substance(s) show  acute toxic/stressful/disrupting action for mitochondria n > m , when testing substance(s) show  a uncoupling action *, like FCCP behaviour, classifiable  as long term toxic action for mitochondria 0,840,860,880,90,920,940,960,9811 2 3 4 5 6 7 8R2 Test 37 °C25 °C      In the first phase, a series of tests for the basal        respiration only were carried out in order to        compare respiration rates and the quality of the        regressions obtained.       Comparing the average value of m in both cases, it       can be seen that at 37 °C mitochondrial         respiration rate is almost 6 times the level         recorded at 25 °C: it is reasonable to assume that        the enzyme systems responsible for the transport        of electrons in the inner mitochondrial membrane                    evolved to perform their functions optimally at a        temperature characteristic of the environment in        which they are immersed (a beef heart cell). At        temperatures lower than optimum, the activity of        these enzymes will decrease and thus the rate at        which reactions they promote take place. Example of a baseline  mitochondrial breath test y = -1,95 x + 3048,84 R2= 0,998 RESPIRATION RATE Ist PHASE - Comparison between 25 °C and 37 °C working temperature Comparison of obtained values highlights that in the case of tests conducted at 25 °C the coefficient of determination has a higher variability and lower values that in the case of tests conducted at 37 °C. In the case of tests conducted at 37 °C, the output signal from the measurement system is more stable and this confirm the hypothesis of mitochondria greater efficiency at this temperature. known toxic compound (positive control) or testing substance(s)  time (1200 sec) O2 content  (A.U.) m respiration rate (negative control) n respiration rate after toxic input 30 mL mitochondria  i.e. [0,75 mg mL-1]fin    FM22 test  highlights       • fast, cheap and easy to handle bioassay • monitoring O2 consumption as DR  • no ATP production  • LC50 determination       Test set-up:   a Clark Oxygen electrode interfaced to a pc   a closed water-jacketed (thermostated at 25 °C) 2.5 mL vessel (reaction chamber)  respiration medium: 0,25 M sucrose, 0,1 M TRIS-HCl pH 7,4   test run 20 min, loading 1200 data.     Bioassay general steps:  beef mitochondria : prepared (Azzone et al., 1979) and stored at -22 °C (standardized method)  blank test : to verify the linear fitting of respiration  pure compound toxicity test : to verify the method sensitivity  internal control for each test : toxicity is quantified comparing the slope (m and n) before and after adding the compound(s)  statistical methodology identified a break-point in the linear fitting (linear fitting of O2 consumption was verified by R2). INTRODUCTION The protocol for the bioassay with the mitochondria of beef heart frozen at -22 °C, (developed by Iero, Manente, Bragadin and Perin, in Chemosphere, 52, 2003) requires that the reaction cell is thermostatically controlled at 25 °C. This value was chosen because it is used as a reference for the state environmental standard (Standard Ambient Temperature and Pressure, SATP). The choice is not, therefore, been supported by assessments on the effectiveness of the test at this temperature, but was dictated by the practice of reporting results to a standard temperature value.  Finally, it was decided to make a comparison between the working temperature of 25 °C and the 37 °C one, chosen as close to cattle body temperature (estimated to be 38.6 °C), then "normal" working temperature for mitochondria extracted from heart.  Improvement on frozen mitochondria bioassay:  a methodological remark. S. Manente,  A. Zuin, A. Iero*, M. Bragadin, G. Ravagnan                                                Molecular Sciences and Nanosystems Dept. Ca’ Foscari University of Venice Dorsoduro, 30123 Venice, Italy *Institute for Applied Ecology University of Canberra Bruce, ACT 2617 Australia IInd PHASE – FM22 toxicity test: comparison between 25 and 37 °C Mitochondria Temperature Toxicity  Test Control Toxicity tests were performed at 37 °C; then, known but variable amounts of toxic compound [e.g. Pb(NO3)2 on the left] were injected at 480 sec to highlight inhibition trend at this temperature and to compare it with obtained values at 25 °C.  The dependence of respiratory inhibition by the concentration of toxic is detectable at both temperatures. They seem to except the values corresponding at the concentration of 20 μM (ΔR = 0.6 at 37 ° C and ΔR = 0 at 25 ° C), but if we look to the pattern of consumption of O2 along time for the tests at 25 ° C we can observe as the index of toxicity is equal to zero because, in these cases, the change in slope between the first and the second part of the curve is not so clear for the macro in Excel® to consider it statistically significant.  Iero A., Manente S., Perin G., Bragadin M. (2003). Frozen mitochondria as rapid water quality bioassay. Chemosphere 53: 1115-1123.  Lee P. N. (1988). Statistics. 377-411 in: Anderson D., Conning D. M. Experimental toxicology. The basic principles. London, Royal Society of Chemistry.  Lehninger A. L. (1979). Biochimica. 2aed. Bologna, Zanichelli.  Ott M., Gogvadze V., Orrenius S. (2007). Mitochondria, oxidative stress and cell death. Apoptosis, 12: 913-922.  Richter C., Gogvadze V., Laffranchi R., Schlapbach R., Schweizer M., Suter M., Walter P., Yaffee M. (1995). Oxidants in mitochondria: from physiology to diseases. Biochimica et Biophysica Acta, 1271: 67-74. Ross S. M. (2003). Probabilità e statistica per l’ingegneria e le scienze. Milano, Apogeo.  Turrens J. F. (2003). Mitochondrial formation of reactive oxygen species. The Journal of Physiology, 552: 335-344.  ESSENTIAL REFERENCES  Running the tests at 37 °C, it can therefore calculate the toxic effect of the compound at concentrations lower than those achievable with the test at 25 °C.  It can be concluded that the bioassay with frozen beef heart mitochondria is more sensitive and accurate at 37 °C.   This reason, together with considerations concerning the rate of respiration higher (due to higher enzyme activity) and increased signal stability, lead to the conclusion that in order to optimize the performance of the test is preferable to control the temperature of the cell reaction at 37 °C.  CONCLUSIONS 02004006008001000120014000 250 500 750 1000 1250 1500[O2] (arbitrary units) Time (s) 00,20,40,60,811,20 50 100 150ΔR Concentration (μM)  Pb(NO3)2 37 °C25 °C20 μM toxic compound at 25 °C REGRESSION COEFFICIENT 

Improvement on frozen mitochondria bioassay: a methodological remark.

Archivio Istituzionale della Ricerca

Name not available

test 
25 °C 37 °C 
m R2 m R2 
1 -0,30 0,967 -1,90 0,999 
2 -0,30 0,976 -2,06 0,998 
3 -0,37 0,991 -1,95 0,998 
4 -0,32 0,992 -1,93 0,998 
5 -0,32 0,985 -1,73 0,997 
6 -0,26 0,954 -1,80 0,998 
7 -0,42 0,987 -1,93 0,996 
8 -0,36 0,896 -2,05 0,998 
average -0,33 0,968 -1,91 0,998 
FM22 end point                 DR = 1 – n/m 
 
n = m , when testing substance(s) don’t show  
acute toxicity for mitochondria 
 
n < m , when testing substance(s) show 
 acute toxic/stressful/disrupting action for 
mitochondria 
n > m , when testing substance(s) show 
 a uncoupling action *, like FCCP behaviour, 
classifiable  as long term toxic action for 
mitochondria 
0,84
0,86
0,88
0,9
0,92
0,94
0,96
0,98
1
1 2 3 4 5 6 7 8
R2 
Test 
37 °C
25 °C
      In the first phase, a series of tests for the basal  
      respiration only were carried out in order to  
      compare respiration rates and the quality of the  
      regressions obtained. 
      Comparing the average value of m in both cases, it 
      can be seen that at 37 °C mitochondrial   
      respiration rate is almost 6 times the level   
      recorded at 25 °C: it is reasonable to assume that  
      the enzyme systems responsible for the transport  
      of electrons in the inner mitochondrial membrane  
                  evolved to perform their functions optimally at a  
      temperature characteristic of the environment in  
      which they are immersed (a beef heart cell). At  
      temperatures lower than optimum, the activity of  
      these enzymes will decrease and thus the rate at  
      which reactions they promote take place. 
Example of a baseline  
mitochondrial breath test 
y = -1,95 x + 3048,84 
R2= 0,998 
RESPIRATION 
RATE 
Ist PHASE - Comparison between 25 °C and 37 °C working temperature 
Comparison of obtained values highlights that in the case of tests 
conducted at 25 °C the coefficient of determination has a higher 
variability and lower values that in the case of tests conducted at 37 °C. 
In the case of tests conducted at 37 °C, the output signal from the 
measurement system is more stable and this confirm the hypothesis of 
mitochondria greater efficiency at this temperature. 
known toxic compound 
(positive control) 
or 
testing substance(s)  
time (1200 sec) 
O
2
 c
o
n
te
n
t  
(A
.U
.)
 
m respiration rate 
(negative control) 
n respiration rate 
after toxic input 
30 mL mitochondria 
 i.e. [0,75 mg mL-1]fin 
 
 
 
FM22 test  highlights  
 
 
 
 
 
• fast, cheap and easy to handle bioassay 
• monitoring O2 consumption as DR  
• no ATP production  
• LC50 determination 
      Test set-up: 
  a Clark Oxygen electrode interfaced to a pc 
  a closed water-jacketed (thermostated at 25 °C) 2.5 mL vessel (reaction chamber) 
 respiration medium: 0,25 M sucrose, 0,1 M TRIS-HCl pH 7,4 
  test run 20 min, loading 1200 data. 
    Bioassay general steps: 
 beef mitochondria : prepared (Azzone et al., 1979) and stored at -22 °C (standardized method) 
 blank test : to verify the linear fitting of respiration 
 pure compound toxicity test : to verify the method sensitivity 
 internal control for each test : toxicity is quantified comparing the slope (m and n) before and after adding the compound(s) 
 statistical methodology identified a break-point in the linear fitting (linear fitting of O2 consumption was verified by R
2). 
INTRODUCTION 
The protocol for the bioassay with the 
mitochondria of beef heart frozen at -22 °C, 
(developed by Iero, Manente, Bragadin and 
Perin, in Chemosphere, 52, 2003) requires 
that the reaction cell is thermostatically 
controlled at 25 °C. This value was chosen 
because it is used as a reference for the state 
environmental standard (Standard Ambient 
Temperature and Pressure, SATP). 
The choice is not, therefore, been supported 
by assessments on the effectiveness of the 
test at this temperature, but was dictated by 
the practice of reporting results to a standard 
temperature value.  
Finally, it was decided to make a comparison 
between the working temperature of 25 °C 
and the 37 °C one, chosen as close to cattle 
body temperature (estimated to be 38.6 °C), 
then "normal" working temperature for 
mitochondria extracted from heart.  
Improvement on frozen mitochondria bioassay:  
a methodological remark. 
S. Manente,  A. Zuin, A. Iero*, M. Bragadin, G. Ravagnan                                                
Molecular Sciences and Nanosystems Dept. 
Ca’ Foscari University of Venice 
Dorsoduro, 30123 Venice, Italy 
*Institute for Applied Ecology 
University of Canberra 
Bruce, ACT 2617 Australia 
IInd PHASE – FM22 toxicity test: comparison between 25 and 37 °C 
Mitochondria 
Temperature 
Toxicity  
Test 
Control 
Toxicity tests were performed at 37 
°C; then, known but variable 
amounts of toxic compound [e.g. 
Pb(NO3)2 on the left] were injected at 
480 sec to highlight inhibition trend 
at this temperature and to compare 
it with obtained values at 25 °C.  
The dependence of respiratory 
inhibition by the concentration of 
toxic is detectable at both 
temperatures. They seem to except 
the values corresponding at the 
concentration of 20 μM (ΔR = 0.6 at 
37 ° C and ΔR = 0 at 25 ° C), but if we 
look to the pattern of consumption 
of O2 along time for the tests at 25 ° 
C we can observe as the index of 
toxicity is equal to zero because, in 
these cases, the change in slope 
between the first and the second 
part of the curve is not so clear for 
the macro in Excel® to consider it 
statistically significant.  
Iero A., Manente S., Perin G., Bragadin M. (2003). Frozen mitochondria as rapid 
water quality bioassay. Chemosphere 53: 1115-1123.  
Lee P. N. (1988). Statistics. 377-411 in: Anderson D., Conning D. M. Experimental 
toxicology. The basic principles. London, Royal Society of Chemistry.  
Lehninger A. L. (1979). Biochimica. 2aed. Bologna, Zanichelli.  
Ott M., Gogvadze V., Orrenius S. (2007). Mitochondria, oxidative stress and cell 
death. Apoptosis, 12: 913-922.  
Richter C., Gogvadze V., Laffranchi R., Schlapbach R., Schweizer M., Suter M., 
Walter P., Yaffee M. (1995). Oxidants in mitochondria: from physiology to 
diseases. Biochimica et Biophysica Acta, 1271: 67-74. 
Ross S. M. (2003). Probabilità e statistica per l’ingegneria e le scienze. Milano, 
Apogeo.  
Turrens J. F. (2003). Mitochondrial formation of reactive oxygen species. The 
Journal of Physiology, 552: 335-344.  
ESSENTIAL REFERENCES 
 Running the tests at 37 °C, it can therefore calculate the toxic effect of the compound at 
concentrations lower than those achievable with the test at 25 °C. 
 It can be concluded that the bioassay with frozen beef heart mitochondria is more sensitive and 
accurate at 37 °C.  
 This reason, together with considerations concerning the rate of respiration higher (due to higher 
enzyme activity) and increased signal stability, lead to the conclusion that in order to optimize the 
performance of the test is preferable to control the temperature of the cell reaction at 37 °C.  
CONCLUSIONS 
0
200
400
600
800
1000
1200
1400
0 250 500 750 1000 1250 1500
[O
2
] 
(a
rb
it
ra
ry
 u
n
it
s
) 
Time (s) 
0
0,2
0,4
0,6
0,8
1
1,2
0 50 100 150
ΔR 
Concentration (μM)  
Pb(NO3)2 
37 °C
25 °C
20 μM toxic compound at 25 °C 
REGRESSION COEFFICIENT 


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Improvement on frozen mitochondria bioassay: a methodological remark.

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Archivio istituzionale della ricerca - Università degli Studi di Venezia Ca' Foscari

Archivio Istituzionale della Ricerca

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